A tumor cell membrane-coated self-amplified nanosystem as a nanovaccine to boost the therapeutic effect of anti-PD-L1 antibody

To improve the response rate of immune checkpoint inhibitors such as anti-PD-L1 antibody in immunosuppressive cancers like triple-negative breast cancer (TNBC), induction of immunogenic cell death (ICD) at tumor sites can increase the antigenicity and adjuvanticity to activate the immune microenvironment so that tumors become sensitive to the intervention of immune checkpoint inhibitors. Herein, a self-amplified biomimetic nanosystem, mEHGZ, was constructed by encapsulation of epirubicin (EPI), glucose oxidase (Gox) and hemin in ZIF-8 nanoparticles and coating of the nanoparticles with calreticulin (CRT) over-expressed tumor cell membrane. EPI acts as an ICD inducer, Gox and hemin medicate the cascade generation of reactive oxygen species (ROS) to strengthen the ICD effect, and CRT-rich membrane as “eat me” signal promote presentation of the released antigens by dendritic cells (DCs) to invoke the tumor-immunity cycle. The biomimetic delivery system displays an amplified ICD effect via Gox oxidation, hydroxyl radical generation and glutathione (GSH) depletion. The induced potent ICD effect promotes DCs maturation and cytotoxic T lymphocytes (CTLs) infiltration, reversing an immunosuppressive tumor microenvironment to an immunoresponsive one. Treatment with the nanosystem in combination with anti-PD-L1 antibody results in distinctive inhibition of tumor growth and lung metastasis, supporting that a potent ICD effect can significantly boost the therapeutic efficacy of the anti-PD-L1 antibody. This self-amplified biomimetic nanoplatform offers a promising means of raising the response rate of immune checkpoint inhibitors.

Introduction
Immunotherapy by employing immune checkpoint inhibitors (ICIs), such as anti-PD-1/L1 antibodies, has revolutionized the paradigm of cancer treatment in the last decade [1,2]. However, the response rate of ICIs is very poor, and about 10-30% has been reported in the cancer with an immunosuppressive microenvironment such as triple-negative breast cancer (TNBC) [3][4][5]. It has been revealed that immunogenic cell death (ICD) can activate the immune microenvironment to potentiate the ICI treatment effect [6]. Tumor cells treated with chemotherapeutic agents anthracyclines such as EPI undergo ICD to release tumor associated antigens and danger associated molecular patterns (DAMPs) including calreticulin (CRT), high mobility group box 1 (HMGB1) and adenosine-5 ′ -triphosphate (ATP) [7,8], reversing an immunosuppressive tumor microenvironment to an immunoresponsive one [9,10]. Encouraging therapeutic outcomes have been reported after treatment with a combination of ICIs with ICD [11,12]. However, ICD induced from one single chemotherapeutic agent is not adequate enough to produce persistent immune responses [13,14].
To address this issue, synergy with multiple intervention methods to amplify the ICD effect has been pursued to boost the therapeutic effect of ICIs [15,16]. The ER stress is often harnessed for strengthening the ICD Peer review under responsibility of KeAi Communications Co., Ltd. effect for potent immunogenic responses. Ca 2+ depletion, hypoxia, and ROS generation have been proposed to effectively escalate the ER stress, thus inducing ICD [17]. Fenton and Fenton-like reactions are often employed for chemodynamic therapy because intracellular ROS could be generated from these reactions under a specific tumor microenvironment [18]. A glucose oxidase (Gox)-based nanozyme system is safe and efficient to induce Fenton reactions in a quite specific and well-controlled way [19], and this nanozyme can oxidize glucose to gluconic acid and hydrogen peroxide (H 2 O 2 ) in tumor cells. Excess glucose consumption may lead to reduced energy supply to tumor cells, which has been utilized for starvation therapy of cancers. Meanwhile, the product H 2 O 2 from glucose oxidation can react with Fe 2+ via the Fenton reaction to generate hydroxyl radicals (⋅OH) and oxidize intracellular glutathione (GSH). A reduced level of GSH is often accompanied with a weakening capacity of scavenging of cytotoxic ⋅OH, thus aggravating the ROS generation and intensifying the ICD effect.
Functionalized nanosized delivery systems have been developed for delivery of therapeutic agents into tumor sites [20,21], and a biomimetic drug delivery system has attracted tremendous attention in recent years [22,23]. Cell membranes from red blood cells [24], platelets [25], natural killer cells [26] and cancer cells [27] have been used for engineering these biomimetic nanoplatforms. Among these cell membranes, the tumor cell membrane can retain the antigens on the cell membrane to evoke immune responses and maintain homotypic adhesion for targeted delivery [28,29]. Besides, CRT on the cell membrane is one of important DAMPs in immunogenic signal transmission, and the presence of CRT promotes engulfment of tumor-associated antigens by antigen-presenting cells (APCs) to activate immune responses. After in vitro treatment with tumor cells to stimulate ICD, CRT can be over-expressed on the cell membrane. The use of the cell membrane with over-expressed CRT could strengthen the immunogenicity [30].
Herein, we constructed a biomimetic nanoparticle to trigger a cascade reaction for ROS generation to amplify the ICD effect and boost the sensitivity of tumor cells to the treatment with anti-PD-L1 antibody. In this system, zeolitic imidazolate framework (ZIF-8) nanoparticles were employed as a drug carrier due to their large surface area, porous interconnectivity and intrinsic pH-induced biodegradability. An ICD inducer EPI, Gox and hemin were encapsulated into ZIF-8 nanoparticles to prepare a drug-loaded nanosystem, EHGZ. This EHGZ system was coated with CRT over-expressed tumor cell membrane to obtain a biomimetic nanoparticle, mEHGZ (Scheme 1A). The tumor cell membrane coating layer on mEHGZ could facilitate their cellular uptake by tumor cells and act as an immune adjuvant. After cellular uptake of the mEHGZ nanoparticles, a Fenton reaction could be triggered by released Gox and hemin from mEHGZ nanoparticles to promote ROS generation and elevate the ER stress, which could amplify the ICD effect in comparison with that induced by EPI alone (Scheme 1B). The released antigens and DAMPs after ICD could function as a nanovaccine to enhance the antigenicity and adjuvanticity, potentiating DCs maturation and cytotoxic T lymphocytes (CTLs) infiltration to activate the tumor immune microenvironment (Scheme 1C). In this context, an enhanced antitumor chemoimmunotherapeutic efficacy could be achieved in a murine 4T1 model. Therefore, the self-amplified biomimetic nanosystem could induce a potent ICD effect and activate the tumor immune microenvironment, boosting the therapeutic efficacy of anti-PD-L1 antibody against tough cancers of TNBC.

Scheme 1.
Construction of a self-amplified biomimetic nanosystem to induce ICD and activate an immune microenvironment for boosting the therapeutic effect of anti-PD-L1 antibody. A) Schematic illustration of the process to prepare mEHGZ nanoparticles by encapsulating EPI, Gox and hemin in ZIF-8 nanoparticles and coating the nanoparticles with the CRT over-expressed tumor cell membrane. B) After endocytosis of mEHGZ nanoparticles into cells, the released EPI from mEHGZ in response to a low pH kills tumor cells in an immunogenic way. Gox and hemin mediate a cascade reaction for generation of hydroxyl free radical (⋅OH) and depletion of intracellular GSH to exert strong stress on the ER, strengthening the ICD effect. C) The released antigens and DAMPs after ICD promote DCs maturation, CD8 + T cells infiltration and cytokines secretion to create an immunosupportive microenvironment to boost the therapeutic effect of anti-PD-L1 antibody.

Cell lines and animals
The 4T1 murine breast cancer cell line and RAW 264.7 cell line were obtained from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). 4T1 cells were cultured in the PRMI 1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin (Hyclone, USA), while Raw 264.7 cells were cultured in DMEM-high glucose medium (Hyclone, USA) with 10% FBS (Gibco, USA) and 1% penicillinstreptomycin (Hyclone, USA) in an incubator with 5% CO 2 at 37 • C. Female mice of 4-6 weeks (18.0 ± 2.0 g) were purchased from Chengdu Dashuo Experimental Animals Co. Ltd. These animals were fed at a specific pathogen free (SPF) standard condition on a 12 h light/12 h dark cycle. All animal experiments were carried out according to the guidelines of the Ethics Committee of West China Hospital, Sichuan University (No. 2018148A and 2018150A).

Synthesis of ZIF-8 and encapsulation of EPI
(1) Synthesis of ZIF-8: ZIF-8 was synthesized by the "one-pot" method in a previous report with modification [31]. The concentrations of zinc acetate and the 2-MI solution were selected by comparing the dynamic light scattering (DLS) size of the ZIF-8 particles shown in Table 1 (supporting information). A zinc acetate aqueous solution (0.25 mL, 200 mg/mL) was dropwise added into 2.5 mL of 2-MI aqueous solution (200 mg/mL). The mixture was stirred at room temperature for 30 min, and then centrifuged (13000 g, 30 min) to obtain white precipitates (ZIF-8).
(2) Encapsulation of EPI: An EPI stock solution (100 μL, 20 mg/mL) was mixed with the zinc acetate solution and the mixture was dropwise added into the 2-MI solution. The same procedure as (1) was repeated to obtain precipitates (EZ). (3) Encapsulation of Gox and hemin: A Gox aqueous solution (50 μL, 20 mg/mL) and hemin (50 μL,10 mg/mL) were mixed with the 2-MI solution. The zinc acetate solution was dropwise added to the prepared mixture. The same procedure as (1) was repeated to obtain precipitates (GHZ). (4) Encapsulation of EPI, Gox and hemin: An EPI stock solution was added to the zinc acetate solution. A Gox aqueous solution and hemin were mixed with the 2-MI solution. The two mixtures were mixed under stirring. The same procedure as (1) was repeated to obtain precipitates (EHGZ).
The loading amounts of EPI and hemin in nanoparticles were determined by fluorescence and UV-vis absorption spectroscopy, respectively. The drug loading efficiency (LE) was calculated by the equation: LE= (m t -m f )/m t , where m t is the total amount of EPI or hemin initially added and m f is its amount in the supernatant.

Collection of tumor cell membrane and coating of membrane onto NPs
4T1 cell membranes were collected via a similar procedure according to the literature [29]. Briefly, cells were collected by scraping after they reached confluence. The collected cells were washed in cold PBS three times, and then in a buffer solution of 30 × 10 − 3 M Tris-HCL (pH = 7.0) with 0.0759 M sucrose and 0.225 M D-mannitol three times. Cells were dispersed in a cocktail solution (1 × ) containing phosphatase inhibitor and protease inhibitor and they were mechanically disrupted via an ultrasonic probe at a power of 35% (3 s on and 5 s off, 20 times) in an ice bath. The solution was centrifuged at 10000 g, 4 • C for 35 min to remove organelles. The collected supernatants were centrifuged at 150000 g, 4 • C for 35 min to obtain the cell membrane. To induce CRT expression on the tumor cell membrane, 4T1 tumor cells were pre-treated with EPI overnight. The above membrane collection process was repeated for those pre-treated cells to obtain the cell membrane with over-expressed CRT. The protein content on the cell membrane was measured via a BCA protein assay kit according to the manufacturer's instruction.
To prepare biomimetic nanoparticles, mEHGZ, the EHGZ nanoparticles were dispersed in DI water. The cell membrane solution with an equal mass of the nanoparticles was added to the nanoparticle solution under stirring. The mixture was disrupted using an ultrasonic probe at a power of 45% (3 s on and 5 s off, 15 times), and the coated nanoparticles were obtained after centrifugation (4 • C, 15000 g, 45 min).

Nanoscale flow cytometry to determine the coating efficiency
4T1 cells were stained with Dio for 15 min to obtain the Dio-staining cell membrane. The Dio-staining cell membrane was collected to coat EHGZ nanoparticles using the same procedure detailed in 2.4. The membrane-coated nanoparticles were subjected to flow cytometry analysis according to the protocol of nanoscale flow cytometry. The coating efficiency was determined from the ratio of Dio + -EPI + nanoparticles to the total number of nanoparticles.

Identification of membrane-associated proteins
The membrane-associated proteins were confirmed via coomassie blue staining and western blotting. The cell membrane collected from 4T1 cells, the cell membrane with over-expressed CRT, or mEHGZ at an equivalent 20 μg of proteins was loaded into each well of a 12% Tris/ glycine SDS-polyacrylamide gel. For coomassie blue staining imaging, gelatin was stained in a coomassie blue fast staining solution for 30 min, and exposed in a gel imaging system. To execute western blotting analysis, proteins were transferred to polyvinylidene fluoride membranes. They were blocked in 5% BSA solution for 1 h, and incubated with an CRT/Na + -K + ATPase antibody overnight at 4 • C and an anti-HRP rabbit antibody for 2 h. The proteins were visualized in an imaging system (Bio Rad Image system).

Cell viability and apoptosis analysis
The in vitro cytotoxicity was evaluated via the CCK-8 assay. 4T1 cells were seeded in 96-well plates for 24 h, and the solutions of EPI, EZ, GHZ, EHGZ or mEHGZ were added at an equal EPI amount of 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2 and 0.1 μg/mL. A PBS solution was used as a control.
Cells were incubated with these solutions for 24 h. The CCK-8 kit in the cell culture medium was added to cells and then incubated for 2 h, the OD 450 value was read to calculate the relative cell viability by normalizing the OD 450 readings of samples by that of the control. Live-dead cell staining was also carried out for direct visualization of the cell viability. 4T1 cells were seeded in 24-well glass plates, and cells were treated with EPI, EZ, GHZ, EHGZ, or mEHGZ at the equivalent EPI concentration of 1 μg/mL for 12 h. Cells treated with PBS were used as a control. Viable cells were stained with calcein as green (λ ex = 494 nm, λ em = 517 nm), while dead cells with DAPI as blue (λ ex = 340 nm, λ em = 488 nm) under a fluorescence microscope.
The apoptosis of 4T1 cells was determined via an Annexin V apoptosis kit via flow cytometry (BD). Briefly, 4T1 cells were seeded in 6-well plates (5 × 10 5 cells) for 24 h, subsequently, they were cultured with EPI, EZ, GHZ, EHGZ, or mEHGZ at the same EPI concentration of 1 μg/mL for 12 h. The treated cells were collected (4 • C, 1000 rpm, 3 min) and suspended in the Annexin V binding buffer, stained and washed according to the supplier's protocol.

Cellular uptake
4T1 cells were seeded in 12-well plates for 24 h and they were then cultured in the medium containing EPI, EZ, GHZ, EHGZ, or mEHGZ at an equivalent EPI concentration of 2 μg/mL for 2 h. They were centrifuged (4 • C, 1000 rpm, 3 min) and washed with PBS for flow cytometry analysis. To visualize the cellular uptake process, cells were seeded in confocal glass dishes and they were subjected to the same treatment. These cells were washed and fixed with 4% paraformaldehyde, and their nuclei were stained with DAPI. The cells were visualized under a confocal scanning laser microscope (CLSM).

Intracellular •OH generation
A ROS probe, 2 ′ , 7 ′ -dichlorofluorescin diacetate (DCFH-DA) (λ ex = 488 nm, λ em = 525 nm), was used to detect in vitro free radicals generation. 4T1 cells were seeded in 24-well plates for 24 h at 37 • C. EPI, EZ, GHZ, EHGZ, or mEHGZ at an equivalent EPI dose of 1.0 μg/mL was added to incubate for 12 h. 1 μM of the DCFH-DA solution was incubated with the treated cell for 30 min. Cells were collected and washed with PBS for flow cytometry analysis. The cell nucleus was stained with DAPI for visualization.

Intracellular glutathione (GSH) detection
To detect the in vitro GSH level, 4T1 cells were seeded in 6-well plates and incubated at 37 • C with 5% CO 2 for 24 h. They were treated with EPI, EZ, GHZ, EHGZ, or mEHGZ at an equivalent EPI dose of 1.0 μg/mL for 12 h. Cells treated with PBS were used as a control. The cells were collected for detecting the intracellular GSH content according to the GSH detection kit's instruction.

Induction of immunologic cell death (ICD)
To confirm ICD of 4T1 tumor cells after treatment with nanoparticles, CRT and HMGB1 were detected via immunofluorescence and the released ATP was analyzed via ATP detection kit. Briefly, 4T1 cells were seeded on a 24-well glass bottom cell culture dish at a density of 1 × 10 5 per well. After 24 h incubation, EPI, EZ, GHZ, EHGZ, or mEHGZ at an equivalent EPI dose of 2.0 μg/mL was added into the well and incubated with cells for 4 h. The cells were washed with PBS twice and fixed with 4% paraformaldehyde. They were incubated with 5% BSA for 30 min, anti-CRT antibody or anti-HMGB1 antibody overnight in 4 • C and APC-conjugated secondary antibody for 2 h. The nuclei were stained with DAPI at room temperature for 8 min. The fluorescence signal of CRT and HMGB1 was detected via a confocal scanning laser microscope (λ ex = 633 nm, λ em = 660 nm). For ATP detection, 1 × 10 5 4T1 cells were seeded on a 6-well plate and cultured with EPI, EZ, GHZ, EHGZ, or mEHGZ. The supernatants were collected, and the extracellular ATP content was measured with an ATP assay kit according to the manufacturer's instruction.

Immune activation effect of CRT-over expressed coated nanosystem
RAW 264.7 cells and bone marrow-derived dendritic cells (BMDCs) were used to evaluate the immune response activated by the collected cell membrane. BMDCs were generated from bone marrow mesenchymal stem cells according to the report [32]. Briefly, BMDCs were obtained from the femurs and tibiae of male C57BL/6 mice at 4-6 weeks. The suspensions were dispersed in a red blood cell lysis buffer and centrifuged to obtain precipitates. The precipitates were incubated in the RPMI 1640 medium containing 10% FBS and 20 ng/mL GM-CSF for 5 days to yield BMDCs. RWA264.7 cells and BMDCs were seeded in 24-well plates and cultured with the cell membrane or the CRT over-expressed cell membranes at the concentration of 0.4, 0.2, 0.1 or 0.05 mg/mL for 12 h. These cells were stained with anti-mouse11c/80 antibodies for flow cytometry analysis. After the obtained BMDCs were cultured in a lower chamber of a Transwell system, 4T1 cells treated with EPI, EZ, GHZ, EHGZ, or mEHGZ at an equivalent EPI dose of 1 μg/mL for 12 h were added into the upper chamber. After 24 h incubation, the BMDCs were collected and stained with anti-11c, anti-80 antibodies for flow cytometry analysis the ratio of CD11c + CD80 + BMDC cells.

In vivo activated immune cells analysis
To examine potent immune responses in 4T1-bearing mice induced by mEHGZ, the percentages of CD8 + T cells and mature DCs were measured via a flow cytometer. The mice were euthanized two days after the last treatment with EPI, EZ, GHZ, EHGZ, mEHGZ, anti-PD-L1 antibody, and mEHGZ + anti-PD-L1 antibody at an equivalent EPI dose of 3 mg/kg, and/or anti-PD-L1 antibody (BE01001) dose of 100 μg.
The tumor, lymph nodes, and spleen were harvested. The tumor was cut into pieces and centrifuged to obtain the supernatant for ELISA analysis of IFN-γ, TNF-α, and IL-6. The pellets were digested in a solution containing hyaluronidase/collagenase IV and Deoxyribonuclease I in a shaker at 37 • C for 30 min to generate a single cell suspension. The cells were filtered through a 70 μm filter and dispersed in 5% BSA to block the non-specific Fc fragment. The isolated cells were stained with fluorochrome-conjugated antibodies: anti-mouse CD45, anti-mouse CD3, anti-mouse CD4, anti-mouse CD8, anti-mouse IFN-γ, anti-mouse CD11c, anti-mouse CD80 or anti-mouse CD86 for flow cytometry analysis. Analysis of CD8 + T cells in the spleen and lymph nodes were executed with a similar procedure as that for the tumor tissue.

Anti-tumor studies in vivo
To establish a 4T1 subcutaneous tumor model, 5 × 10 5 4T1 cells were subcutaneously injected into the right lower back of mice. The tumor volume (V) was monitored and calculated from the equation: V = ab 2 /2, where a and b are the length and width of the tumor. When the tumor volume reached about 50-75 mm 3 , the mice were randomly divided into eight groups (n = 5 in each group) and they were treated with saline, free EPI, EZ, GHZ, EHGZ, mEHGZ, anti-PD-L1 antibody, and mEHGZ + anti-PD-L1 antibody. These formulations were intratumoral injection at an equivalent EPI dose of 3 mg/kg. An anti-PD-L1 antibody (BE01001) at a dose of 100 μg was intraperitoneally injected on the following day. This treatment cycle was repeated three times every two days. The tumor volume and the body weight were recorded every other day. At the end of the treatment, mice were sacrificed to collect tumors and major organs for H&E, TUNEL, KI67 staining, and immunohistochemistry analysis.

Statistical analysis
All quantitative values were presented as means ± SD. Student's ttest was used for the statistical analysis of differences between two groups, and statistical significance was set at a 95% confidence level for all tests, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Synthesis and characterizations of biomimetic mEHGZ nanoparticles
The process for self-assembly of nanoscale metal-organic frameworks (nMOFs) from Zn 2+ and 2-MI, encapsulation of EPI, Gox and hemin into nMOFs, and coating of the nanoparticles with tumor cell membrane is presented in Scheme 1. The ZIF-8 nanoparticles with porosity display an excellent encapsulation ability, and the EPI and hemin entrapment efficiency in ZIF-8 nanoparticles are 81.42% and 84.7%, respectively. (Figs. S1 and S2). Transmission electron microscopy (TEM) images (Fig. 1A) show that these nanoparticles display a regular tetrahedral shape with a diameter size of 110 nm. The thickness of the outermost layer composed of the tumor cell membrane is about 10 nm. The hydrodynamic size of EHGZ via DLS is 123.66 ± 2.83 nm. After coating the cell membrane onto EHGZ, the DLS size increases to 133.92 ± 2.93 nm (Fig. 1C), which is consistent with the TEM result. Furthermore, the surface charge after coating changes from 17.77 ± 0.95 mV to -26.47 ± 1.98 mV (Fig. 1D), which indicates successful coating of the cell membrane onto EHGZ nanoparticles. Powder X-ray diffraction (XRD) patterns (Fig. 1E) of EZ, GHZ, EHGZ, mEHGZ are identical to those of blank ZIF-8 nanoparticles, which suggests that the cargo in the cavity of ZIF-8 nanoparticles and the sonication process for facilitating the coating process have no impact on the crystallinity of ZIF-8. Fourier transform infrared (FTIR) spectra support EPI, Gox and hemin are encapsulated in ZIF-8 nanoparticles (Fig. S3). It has been reported that ZIF-8 nanoparticles degrade at a low pH and they are a natural pHresponsive nanocarrier to release the loaded drug in a controlled manner [33]. The in vitro release profile for EPI from ZIF-8 nanoparticles confirms that these nanoparticles are responsive to a low pH. The released amount of EPI in a buffer at pH 5.4 is about ten times higher than that in a buffer at pH 7.4 (Fig. 1F). A similar release result for mEHGZ is observed, and the drug release rate at pH 5.4 is quicker than that at pH 7.4 (Fig. S4). The sustained drug release from mEHGZ lasts up to 168 h, and at this time point the drug carrier is observed to be cleared through the liver metabolism. The detailed information of the metabolism process of ZIF-8 nanoparticles is shown in Table 2 (supporting  information). Furthermore, there are negligible changes in the hydrodynamic size distribution and polydispersion index (PDI) of EHGZ and mEHGZ in PBS for 72 h (Fig. S5), suggesting these nanoparticles are favorably stable.
After tumor cells are pre-treated with a chemotherapeutic drug like EPI, ICD may be triggered in these cells and CRT could migrate from the ER to the cell membrane. The collected cell membrane for coating EHGZ nanoparticles would evoke the immune system because CRT may bind to CD91 on APCs [34]. After 4T1 tumor cells were treated with 2 μg/mL EPI for 12 h, significantly CRT is seen to relocate on the tumor cell membrane (Fig. 1G and Fig. S6) according to western blot analysis. The membrane-associated protein profiles in the coomassie blue staining images of the CRT over-expressed membrane are identical to those of mEHGZ (Fig. 1H), suggesting the membrane proteins are intact retained on the mEHGZ nanoparticles. Furthermore, the EDS mapping supports that the membrane is successfully coated onto the EHGZ nanoparticles since the P element is the main component of cell membrane proteins (Fig. 1B). As shown in Fig. S7, after the cell membrane was stained with Dio, it emits green fluorescence (λ ex = 484 nm, and λ em = 501 nm). The ratio of the cell membrane coated onto the nanoparticles was evaluated via nanoscale flow cytometry. According to Fig. 1I, the membrane coating efficiency is 11.8%, while it raises nearly two folds to 21.0% after centrifugal collection. The coating efficiency is similar to that reported by Liu et al. [35]. However, the membrane coating efficiency is quite low via the sonication approach. The membrane coating method need to be optimized for a high coating efficiency to maximize the use of the tumor cell membrane.

In vitro cytotoxicity and cell uptake of mEHGZ
It is reported that the proteins on the cancer cell membrane play an important role in homologous recognition [36,37], therefore cancer cell membrane-camouflaged nanoparticles may have the potential to achieve specific targeting towards the same cancer cells. As shown in the CLSM images ( Fig. 2A), after incubation of free EPI, EHGZ, and mEHGZ with 4T1 cells for 2 h, the red fluorescence intensity in 4T1 cells incubated with mEHGZ is much stronger than that of the cells treated with free EPI, suggesting that mEHGZ promotes cellular uptake in 4T1 cells. Furthermore, the cellular uptake efficiency is 62.33%, 82.4% and 91.04% for free EPI, EHGZ, and mEHGZ, respectively (Fig. 2B), which is coincident with CLSM images. The EHGZ nanoparticles display an enhanced uptake efficiency since these positively charged nanoparticles may interact with negatively charged tumor cell membrane. mEHGZ nanoparticles outperform free EPI and EHGZ in their cellular uptake since the coated cell membrane may promote homologous and rapid fusion with the target tumor cells. Furthermore, the uptake efficiency of mEHGZ by 293T cells, HUVEC and RAW264.7 cells is 4.71, 3.49, 4.76 folds lower than that by 4T1 cells (Fig. S8), which suggests that the coated 4T1 cell membrane could medicate homotypic target ability.
The CCK-8 assay was used to evaluate the cell viability of 4T1 cells after incubation with EHGZ and mEHGZ nanoparticles. As shown in Fig. 2C, when EPI, Gox and hemin were loaded into ZIF-8 nanoparticles, the lowest cell viability is observed in 4T1 cells, indicating that Gox may exert a starvation effect on tumor cells and hemin promotes the Fenton reaction and GSH depletion, synergistically reducing the cell viability. mEHGZ displays negligible cytotoxicity against APCs such as RAW264.7 and DC2.4 cells (Fig. S9), which supports that mEHGZ could not influence the viability of APCs. Furthermore, cytotoxicity induced by the selfamplified delivery system was confirmed with a live and dead viability assay kit. Exposure to EHGZ and mEHGZ leads to the highest ratio of dead cells stained by DAPI compared with other groups (Fig. 2E). The 4T1 cells death mechanism after different treatments was studied via an Annexin V-FITC apoptosis kit and a flow cytometer. According to Fig. 2F, the percentage in the Q2 of the flow cytometry for late apoptosis is 18.1%, 35.4%, 54.8%, 72.4%, and 86.0% after treatment with EPI, EZ, GHZ, EHGZ, and mEHGZ, respectively. mEHGZ nanoparticles have the highest potency in inducing apoptosis of 4T1 cells in comparison with other groups at an equal dose of EPI. These consistent results from the CCK-8 assay, live and dead staining and the apoptosis assay suggest that mEHGZ could be an excellent therapeutic nanomedicine against 4T1 tumor cells.

ROS generation and depletion of GSH by mEHGZ nanoparticles
Intracellularly generated ROS, a competent inducer for ICD, perturb the balance between protein folding load and capacity, resulting in an elevated ER stress [38]. Compared to normal cells, GSH, a powerful reductant, is over-expressed in tumor cells to maintain the oxidation-reduction homeostasis in a tumor microenvironment [39,40]. We designed a cascade reaction to promote ROS generation and deplete intracellular GSH to strengthen the ER stress that could amplify the ICD effect. After EPI, Gox and hemin were released from the porous ZIF-8 nanoparticles, Gox oxidizes glucose to hydrogen peroxide, and Fe 2+ in hemin reacts with hydrogen peroxide to generate cytotoxic ⋅OH and Fe 3+ . The generated Fe 3+ oxidizes GSH, leading to an up-regulated ROS level and a depleted level of GSH, thus breaking the oxidation-reduction balance (Fig. 3A). A ROS probe, DCFH-DA, is non-fluorescent, but it can be oxidized by intracellular ROS to produce green fluorescent 2 ′ , 7 ′ -dichlorofluorescein (DCF). Thus, the probe was used to evaluate the ROS level inside tumor cells via flow cytometry (Fig. 2D) and fluorescence microscopy. It can be seen that the fluorescence intensity in cells treated with EHGZ or mEHGZ is remarkably strong, while weak fluorescence is seen in cells treated with EZ or GHZ nanoparticles, which  indicate that treatment with EPI combined with Gox and hemin could generate a high level of ROS in tumor cells (Fig. 2G). The ROS generation was detected via mice ROS ELISA detection kit in the tumor tissue. The ROS content increases from 43.25 IU/mL to 104.62 IU/mL in the tumor tissue after treatment with EPI or mEHGZ (Fig. S10). Furthermore, the GSH level in 4T1 cells after treatment with mEHGZ reduces 1.7 folds compared to the EPI-treated group (Fig. 3B) and a 2.2-fold reduction in the GSH level is seen in the tumor tissue after treatment with mEHGZ (Fig. S11), confirming the self-amplified nanoparticles could reduce the intracellular GSH level.

In vitro ICD induced by nanoparticles
CRT exposure, HMGB1 transfer and ATP release are the hallmarks of ICD. As shown in Fig. 3C, the concentration of ATP in the supernatant of cells after treatment with mEHGZ is 250.30 μM, in comparison with 91.40 μM in the group treated with EPI. An 2.74-fold increase in the released ATP indicates the amplified ER stress could strengthen the ICD effect compared to a single chemotherapeutic drug EPI. Meanwhile, the ATP content in tumor tissue is 48.29 nM and 103.59 nM in the tumor tissue after treatment with EPI and mEHGZ, respectively (Fig. S12). The majority of HMGB1 is located in the cell nucleus after PBS treatment, but it gradually transfers from the nucleus to the cytoplasm in the groups treated with EPI, EZ, GHZ, or EHGZ nanoparticles. Strikingly, the vast majority of HMGB1 is found in the cytoplasm in the cells treated with mEHGZ (Fig. 3D). CRT exposure on the cell membrane triggers DCs to swallow the released antigens, which invokes the tumor-immunity cycle. CLSM images in Fig. 3E show that cells treated with EPI alone leads to a negligible level of CRT exposure, while the strongest red fluorescence intensity is seen in the mEHGZ-treated group, suggesting that mEHGZ can strongly induce CRT transportation from the ER to the cell membrane. These DAMPs results suggest a potent ICD effect is induced by the self-amplified delivery system.

Cell membrane with overexpressed CRT as an immune adjuvant
CRT exposure on the cell membrane activates DCs to present and process antigens, acting as a promising immune adjuvant [41,42]. The function of the cell membranes with over-expressed CRT was evaluated after culture of the cell membrane with BMDCs and RAW 264.7 cells. The proportion of matured cells is found to be positively correlated with the concentration of the cell membrane ( Fig. S13 and Fig. S14). The ratio of CD80 + BMDCs is 51.03% when they were co-cultured with 0.2 mg/mL of the CRT-overexpressed cell membrane, while it is 34.8% after treatment with the normal 4T1 cell membrane at the same concentration (Fig. S13). The membranes with over-expressed CRT at a concentration of 0.2 mg/mL is able to induce 56.83% of RAW 264.7 cells to become mature, while only 27.17% is seen after treatment with the normal cell membrane at the same concentration (Fig. S14), indicating that CRT acts an important player to promote APCs maturation.
To mimic the in vivo ICD effect, BMDCs were inoculated in the lower chamber of a Transwell chamber and 4T1 cells were placed in the upper chamber after they were treated with nanoparticles for 12 h (Fig. 4A). Both cells were incubated for 24 h, it can be seen that the maturation rate of BMDCs in the mEHGZ-treated group is 33.3%, three times of that in the EPI-treated group (Fig. 4B), indicating that mEHGZ-treated 4T1 cells release a high level of tumor-related antigens and DAMPs to induce maturation of BMDCs.

In vivo immune activation
The released tumor-associated antigens and DAMPs after ICD of tumor cells promote DCs maturation and CTLs infiltration, activating the tumor immune microenvironment. Anti-PD-L1 antibody can bind to PD-1 expressed on T cells to mediate the "immune escape" [43], but its therapeutic effect is not very potent against tumor cells, especially those with an immunosuppressive microenvironment. It has been demonstrated that anti-PD-L1 antibody in combination with ICD can enhance its tumor-killing effect [44,45]. DCs present and process antigens to T cells to activate the innate and adaptive immune, and DCs maturation and cytokines secretion are two essential indicators for T cell activation and the cell-killing therapeutic effect. Mice were treated three times with EPI, EZ, GHZ, EHGZ, mEHGZ, anti-PD-L1 antibody, and mEHGZ + anti-PD-L1 antibody, they were sacrificed to analyze the immune-relevant cells three days after the last treatment.
The percentage of mature DCs, CD11c + CD80 + CD86 + , is 36.1% after treatment with mEHGZ + anti-PD-L1 antibody, compared with 15.4% in the EPI-treated group, 17.1% in the EZ-treated group, 13.7% in the GHZtreated group, 20.7% in the EHGZ-treated group, and 14.9% in the group treated with the antibody alone ( Fig. 4C and Fig. S15), supporting that the treatment with mEHGZ and anti-PD-L1 antibody induces a significant level of DCs maturation in tumors. A similar result of promoting DCs maturation is found in lymph nodes after treatment with mEHGZ + anti-PD-L1 antibody (Fig. S16). These encouraging results can be ascribed to synergistic action of mEHGZ and the antibody. mEHGZ is able to induce ICD to evoke strong immune responses, and potent ICD is supported with a high level of CRT exposure on the cell membrane on the tumor slices of the treated mice (Fig. 4D). The anti-PD-L1 antibody is able to block the PD-L1 and PD-1 axis to enhance the chemotherapeutic effect. Cytokines play an essential role in eliciting immune responses [46]. For example, IFN-γ is a key mediator of Th1 cells [47], and TNF-α regulates the function of immune cells [48]. According to the ELISA assay results (Fig. 4E-G), the IFN-γ concentration is about 263.50 pg/mL in the mice group treated with mEHGZ, and it is 129.23 pg/mL in the EPI-treated group, while it increases to 380.44 pg/mL when the mice were synergistically treated with mEHGZ and the antibody, suggesting robust T cell-mediated immune responses are elicited. Meanwhile, TNF-α and IL-6 in the mEHGZ + anti-PD-L1 antibody treated group are upregulated and their concentrations are 2.5 and 6.4 folds of those in the EPI-treated group.
CD8 + T cells are the main workforce in the immune system to battle with tumors, and the number of CD8 + T cells is closely associated with the efficiency of inducing immune responses (Fig. 5A) [49]. The percentage of CD8 + T cells in the tumor, lymph node, and spleen were analyzed, and the ratios in these tissues are presented in Fig. 5B-E (gating strategies are shown in Figs. S17 and S18). The CD8 + T cell ratio increases from 1.83% in the EPI-treated group to 33.0% in the group treated with mEHGZ + anti-PD-L1 antibody, and this combinational treatment results in nearly 18-fold amplification of the number of CD8 + T cells in the tumor ( Fig. 5B and C). A similar increase in the percentage of CD8 + T cells in the spleen and lymph nodes confirms the immune system is dynamically evoked (Figs. S19 and S20). The percentage of CD8 + T cells in the spleen and lymph nodes is 10.5% and 16.5% in the mice treated with EPI, respectively, and it increases to 44.0% and 45.4% in the group treated with mEHGZ + anti-PD-L1 antibody ( Fig. 5D and E). These results suggest that mEHGZ could induce ICD to release antigens, and these antigens are effectively engulfed by DCs and then presented to T cells to activate the immune system for enhancement in CTLs infiltration into the tumor. The immunofluorescence slices also support that the number of CD8 + T cells infiltrated into the tumor is distinctively upregulated (Fig. 5F). After blocked CD8 + T cell via intraperitoneal injection anti-CD8 antibody, the therapeutic effect of mEHGZ combination with anti-PD-L1 was weaker than in the immunocompetent mice (Fig. S21), showing CD8 + T cells play important role in killing tumor cells. The treatment with mEHGZ+anti-PD-L1 antibody efficiently triggers the infiltration of IFN-γ + CD8 + T cells into the tumor. 31.2% of IFN-γ + CD8 + T cells is seen after the combinational treatment, which is 11.5 folds higher than that in the EPI-treated group (Fig. 5G and H). These results confirm that the synergistic treatment with mEHGZ and anti-PD-L1 antibody could induce vigorous in vivo adaptive immune responses.  = 50 μm). G) H&E staining of lung tissues at the end of the anti-tumor experiment (scale bar = 100 μm). Data are represented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Anti-tumor therapeutic effect of mEHGZ nanoparticles
4T1-bearing mice were used to evaluate the in vivo anti-tumor effect of mEHGZ nanoparticles. EPI, EZ, GHZ, EHGZ, and saline were used as controls. After treatment of the mice three times every two days (Fig. 6A), EPI treatment barely suppresses tumor growth, while intervention with EHGZ and mEHGZ nanoparticles leads to pronounced delay in tumor growth, which may be ascribed to the elevated ER stress and the amplified ICD effect. Treatment with anti-PD-L1 antibody does not have an obvious effect on inhibition of tumor growth, while remarkable shrinkage of tumors is seen in mice after administration of anti-PD-L1 antibody combined with mEHGZ nanoparticles (Fig. 6B), which indicates that induced tumor cell death as well as an activated tumor immune microenvironment by mEHGZ nanoparticles could favorably potentiate the treatment effect of anti-PD-L1 antibody. Tumors were harvested and weighed at the end of the treatments. The tumor weights are consistent with the tumor volumes and the tumor in the group treated with mEHGZ + anti-PD-L1 antibody is the lightest, confirming this combinational treatment could effectively inhibit tumor cell proliferation (Fig. 6C). Treatment with mEHGZ and the anti-PD-L1 antibody displays the highest tumor growth inhibition rate of 82.02%, while 36.41% in the group after EPI treatment (Fig. S22). There was no significant body weight change during treatment with EHGZ or mEHGZ (Fig. 6D), indicating that negligible systemic toxicity of the constructed nanosystem. However, the EPI-treated mice lose their weights since day 16 due to the adverse effect induced by this therapeutic agent. There are no obvious pathologic changes in H&E staining slices of main organs, such as heart, liver, spleen and kidney, in mice treated with EHGZ, mEHGZ, mEHGZ + anti-PD-L1 antibody, suggesting that the selfamplified delivery system has excellent safety and biocompatibility (Fig. S23). Meanwhile, the remarkable treatment effect is also confirmed from the TUNEL and KI67 staining tumor sections ( Fig. 6E and F). The tumor tissue in the group treated with mEHGZ + anti-PD-L1 antibody contains the highest ratio of dead cells and the KI67 signal is the weakest, suggesting significant apoptosis of tumor cells and severe damage of tumor cells proliferation. Impressively, metastatic nodules in the hematoxylin-eosin (H&E) staining lung sections are barely detectable in the groups treated with mEHGZ or mEHGZ + anti-PD-L1 antibody since induced potent immune responses prevent pulmonary metastasis, while lung metastasis are seen in these groups treated with EPI, EZ and GHZ, which are circled in Fig. 6G. Furthermore, mEHGZ displays a potent anti-tumor effect in the CT26-bearing mice (Fig. S24). The tumor growth weight is reduced from 3449.54 mg in the blank group to 1020.34 mg and 416.24 mg after treatment with EPI and mEHGZ, respectively. Therefore, mEHGZ could be a promising nanosystem for effectively inducing ICD to activate the tumor immune microenvironment and boost the therapeutic effect of anti-PD-L1 antibody.

Conclusion
In summary, we have successfully engineered a self-amplified biomimetic nanosystem, mEHGZ, in which EPI, Gox and hemin were loaded into ZIF-8 nanoparticles and the nanoparticles were coated with tumor cell membrane with over-expressed CRT. Homologous recognition of tumor cell membrane facilitates a high uptake efficiency of mEHGZ nanoparticles by 4T1 cells. CRT on the cell membrane of mEHGZ nanoparticles as an immune adjuvant promotes BMDCs and RAW 264.7 cells maturation. After endocytosis, the released EPI as an ICD inducer from mEHGZ in response to a low pH stimulates tumor cell death, releasing antigens and DAMPs to enhance the immunogenicity. Gox oxidizes glucose to reduce energy supply to tumor cells as well as produce H 2 O 2 that reacts with co-released hemin to generate cytotoxic •OH and deplete intracellular GSH. The excess generated ROS exert a great level of stress to the ER and the ICD effect is significantly strengthened, which is supported with CRT exposure on the cell membrane, HMGB1 transfer to the cytoplasm and ATP release. This potent ICD induces DCs maturation and CD8 + T cells infiltration to the tumor tissue for adaptive immune responses, which creates an immune-activated microenvironment. In this microenvironment, tumor cells become sensitive to the treatment with anti-PD-L1 antibody, and effective inhibition of tumor growth and restricted tumor lung metastasis are achieved in a 4T1 tumor-bearing mice model after treatment with an anti-PD-L1 antibody and mEHGZ. Thus, this nanosystem could be a promising nanoplatform to act as a nanovaccine to enhance the antigenicity and adjuvanticity and boost the treatment potency of anti-PD-L1 antibody for cancers including TNBC.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Resistance to Obesity in SOD1 Deficient Mice with a High-Fat/High-Sucrose Diet

Metabolic syndrome (Mets) is an important condition because it may cause stroke and heart disease in the future. Reactive oxygen species (ROSs) influence the pathogenesis of Mets; however, the types of ROSs and their localization remain largely unknown. In this study, we investigated the effects of SOD1, which localize to the cytoplasm and mitochondrial intermembrane space and metabolize superoxide anion, on Mets using SOD1 deficient mice (SOD1−/−). SOD1−/− fed on a high-fat/high-sucrose diet (HFHSD) for 24 weeks showed reduced body weight gain and adipose tissue size compared to wild-type mice (WT). Insulin secretion was dramatically decreased in SOD1−/− fed on HFHSD even though blood glucose levels were similar to WT. Ambulatory oxygen consumption was accelerated in SOD1−/− with HFHSD; however, ATP levels of skeletal muscle were somewhat reduced compared to WT. Reflecting the reduced ATP, the expression of phosphorylated AMPK (Thr 172) was more robust in SOD1−/−. SOD1 is involved in the ATP production mechanism in mitochondria and may contribute to visceral fat accumulation by causing insulin secretion and insulin resistance.

Introduction
Metabolic syndrome (Mets) is a condition in which visceral obesity combined with hypertension, hyperglycemia, and abnormal lipid metabolism predisposes to cardiovascular disease and is closely related to insulin resistance [1]. Mets is a complex syndrome because it is related to many organs, including the heart, liver, skeletal muscle, and adipose tissue, and the exact pathogenesis is still largely unknown [2]. Several reports have suggested that oxidative stress is involved in the pathogenesis of Mets [3,4], and oxidative stress levels are reported to be increased in human obesity and decreased with weight loss [5]. Oxidative stress is a condition in which reactive oxygen species (ROSs) are produced beyond their antioxidant capacity. Excessive amounts of ROSs that cannot be removed by the antioxidant system attack and damage proteins, lipids, and nucleic acids [6]; however, physiological levels of ROSs have essential roles in biological reactions [7].
AMP-activated protein kinase (AMPK) functions as a metabolic sensor and plays a vital role as a significant regulator in cellular energy homeostasis [8]. AMPK is implicated in metabolic syndrome, and its pathogenesis, insulin resistance [9,10], and ROSs are known to modulate AMPK signaling.
This study focused on the superoxide anion (O 2 · − ) among the ROSs. O 2 · − is produced by the leak of electrons under oxygen in the mitochondrial electron transport chain, xanthine oxidase, the cytochrome p450 monooxygenases, lipoxygenase, nitric oxide synthase, and the NADPH oxidase [11]. O 2 · − is slowly dismutated to hydrogen peroxide (H 2 O 2 ) and molecular oxygen spontaneously (rate constant = 8 × 10 4 /mol/s); however, superoxide dismutase (SOD) could rapidly dismutate O 2 · − (rate constant = 2 × 10 9 /mol/s). The action of ROS may be working locally, and local kinetics should be considered [12]. Mitochondrial ROSs has been reported to activate AMPK secondary to altering the ADP/ATP ratio [13]. Therefore, we propose that O 2 · − acts at the mitochondrial localization and contributes to metabolic syndrome's pathogenesis via AMPK. Three types of SOD are known in mammalian cells. Among them, the manganese SOD (SOD2) present in the mitochondrial matrix and the copper-zinc SOD (SOD1) in the mitochondrial intermembrane space. SOD1 is also localized in the cytoplasm [11]. ATP sensed by AMPK is formed in the mitochondrial electron transfer system. The O 2 · − formed by electrons leaking into the mitochondrial intermembrane space during this process is thought to be metabolized by SOD1. In this study, we investigated the role of SOD1 in the pathogenesis of Mets. Namely, systemic SOD1-deficient mice (SOD1 −/− ) were fed on a high-fat/high-sucrose diet (HFHSD) [14,15] and were compared with wild-type mice (WT).

Animal, Genotyping, and Diets
Male SOD1 −/− mice were obtained from Jackson Laboratory (Bar Harbor, Maine). These mice were backcrossed to C57BL/6J strain mice for eight generations as we did previously [16]. Next, we interbred heterozygous SOD1 +/− mice to obtain wild-type (WT) mice and homozygous SOD1 −/− mice within the same litter. Animals were housed in the facility on a 12-h light-dark cycle in a temperature-controlled room. The male SOD1 −/− and SOD1 +/+ (WT) as control littermates were fed on either a standard diet (ND; CE-7; CLEA Japan, Inc. Tokyo, Japan) or a high-fat/high-sucrose diet (HFHSD; F2HFHSD with 28.3% of calories from carbohydrates, 54.5% from fat, and 17.2% from protein; Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum from 6 weeks of age for 24 weeks (Table 1). We made four groups (WT fed on ND, SOD1 −/− fed on ND, WT fed on HFHSD, SOD1 −/− fed on HFHSD). Body weights were recorded every two weeks. Genotyping for the SOD1 gene (accession number: NM_011434, XM_128337 and XM_358882) was performed by polymerase chain reaction analysis using genomic DNA isolated from the tail tip using the following primers.

Tissue Preparation and Histology
Mice were anesthetized deeply with isoflurane and sacrificed. Blood was collected, and organs were perfused with Krebs' Ringer bicarbonate solution (NaCl 118.3 mmol/L, KCl 4.7 mmol/L, CaCl 2 2.5 mmol/L, MgSO 4 1.2 mmol/L, KH 2 PO 4 1.2 mmol/L, NaHCO 3 25 mmol/L, D-glucose 5.5 mmol/L). The weight of liver, epididymal fat, and pancreas were measured, and liver, epididymal fat, aorta with fat tissue, and pancreas were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned. These samples were stained with hematoxylin and eosin. Images were obtained with a microscopic system (BZ-X710; Keyence, Osaka, Japan). The cell size of adipocytes was measured with Image J.

Measurement of Metabolites
Serum levels of triglyceride, total cholesterol, and ALT were analyzed by enzymatic assays (FUJIFILM Wako Pure Chemical, Osaka, Japan). Blood glucose levels were determined using a blood glucose monitoring system, FreeStyle (Nipro, Osaka, Japan). Serum insulin levels were measured using ELISA kits (FUJIFILM Wako Shibayagi, Gunma, Japan). HOMA-IR as insulin resistance was calculated by multiplying fasting glucose level by fasting serum insulin level. An oral glucose tolerance test (OGTT) was performed after 12 h of fast. D-Glucose 3 g/kg body weight was orally administered. The vein blood was collected at 0, 15, 30, 60, and 120 min after glucose injection in OGTT.

Islet of Pancreas and Beta Cell
The weight of the pancreas was calculated from the weight of the β-cells using the method previously described [17,18]. The pancreas was stained with hematoxylin and eosin. Islet area per pancreatic area was calculated for three consecutive sections, and the average of the area percentage was defined as [islet area]/[pancreas area] (%). Then, double immunofluorescence staining of the pancreas was performed with both insulin and glucagon as beta cells and alpha cells of the islet, respectively. The tissue on the slides was permeabilized with 0.1% Triton X-100, and non-specific binding sites were blocked with 0.5% BSA. The slides were incubated overnight at 4 • C with both primary antibodies (anti-insulin antibody; 1:800 dilution; Cell Signaling Technology, Danvers, MA, USA; Cat#8138, anti-glucagon antibody; 1:400 dilution; Cell Signaling Technology; Cat#8233). After incubation, the slides were washed and incubated for 3 h at 37 • C with corresponding Alexa Fluor-488 (1:500 dilution; Cell Signaling Technology; Cat#4408) and Alexa Fluor-594 antibodies (1:250 dilution; Cell Signaling Technology; Cat#8889). The slides were washed three times and mounted under glass coverslips with Vectashield Antifade Mounting Medium containing DAPI for nuclear identification (Vector Laboraryories, Burlingame, CA, USA). Images were obtained with a BZ-X710 microscope (Keyence, Osaka, Japan). The area was calculated using the provided analyzing software. The area of each insulinand glucagon-expressing cell was measured in three consecutive sections.

Oxygen Consumption and Food Intake
To assess oxygen consumption, carbon dioxide, and intake of food per day, each mouse was placed in each metabolic chamber system for 24 h after two days for getting (Columbus Instruments, Columbus, OH, USA). The food box with the same diet was kept in the metabolic chamber. The intake of food was calculated by subtraction of the weight after the experiment from the weight before. The diet and water were supplied ad libitum during the experiment.

Measurement of Skeletal Muscle and Vascular Superoxide Production
The superoxide production of aortic rings was assessed with dihydroethidium staining (DHE) (Thermo Fisher Scientific, Waltham, MA, USA), as previously described [19]. Skeletal muscles and aortic rings were snap-frozen with liquid nitrogen and embedded in an optimal cutting temperature medium (Sakura Finetek Japan, Tokyo, Japan). The frozen sections were immediately cut into 10-µm-thick sections and mounted on glass slides. Samples were then incubated at room temperature for 30 min with DHE (2 × 10 −6 mol/L) and protected from light. Images were obtained with a microscopic system (BZ-X710; Keyence) with an excitation wavelength of 540 nm and an emission wavelength of 605 nm. The fluorescence intensity of DHE staining was measured using analyzing soft (Keyence).

ATP Level and ADP/ATP Ratio of Skeletal Muscle
Mice were anesthetized deeply with isoflurane and sacrificed. Immediately gastrocnemius muscles were collected and kept in liquid nitrogen. The muscles were homogenized with a proprietary buffer in the assay kit. ATP and ADP/ATP ratio was measured with a commercial kit, EnzyLight ATP assay kit, and ADP/ATP ratio assay kit (BioAssay Systems, Hayward, CA, USA).

Statistical Analysis
Results are shown as means ± SEM. The Kolmogorov-Smirnov test was used to test normality. Data for body weights, OGTT, and vascular relaxation were analyzed by 2-way ANOVA with repeated measures followed by the post hoc test with Bonferroni correction for multiple comparisons. The other data were analyzed by 1-way ANOVA followed by the post hoc test with Tukey's multiple comparison procedure. All statistical analyses were performed with GraphPad Prism Software version 7 (GraphPad Software; La Jolla, CA, United States). Differences were considered significant if p < 0.05.

Study Design and Changes with ND or HFHSD
WT and SOD1 −/− were divided into the normal diet (ND) and high-fat/high-sucrose diet (HFHSD) groups, respectively, for a four-group study; the HFHSD group received HFHSD for a total of 24 weeks from 6 to 30 weeks of age ( Figure 1A). Western blot analysis of skeletal muscle showed similar expression of SOD2 but not SOD1 in SOD1 −/− compared to WT ( Figure 1B). Dihydroethidium (DHE) (blue fluorescence) is converted to 2-hydroxyethidium (ex 500-530 nm/em 590-620 nm) (red fluorescence) by O 2 · − , so DHE staining was used to detect O 2 · − in skeletal muscle. SOD1 −/− could not dismute O 2 · − , so red fluorescence was observed in the cytoplasm; in HFHSD, red fluorescence was enhanced even in WT, and the most robust red fluorescence was observed in SOD1 −/− fed on HFHSD ( Figure 1C).
Weight gain of SOD1 −/− fed on HFHSD was blunted compared to WT and comparable to ND-fed mice despite the HFHSD ( Figure 1D). To investigate the factors that contributed to the lack of weight gain in SOD1 −/− , the degree of lipid storage in the organs was measured. Epididymal fat, liver, and periaortic fat were evaluated. The total size of epididymal fat from SOD1 −/− was smaller than WT fed on HFHSD, and the adipocyte size of SOD1 −/− was also smaller than WT in HFHSD, detected by hematoxylin and eosin (HE) stain ( Figure 1E). Adipocyte size in periaortic fat was likewise smaller in SOD1 −/− than WT fed on HFHSD; even when treated with ND, it was significantly smaller in SOD1 −/− than WT ( Figure 1F). HE staining of the liver showed fat deposition in HFHSD but to a lesser extent in SOD1 −/− . Serum alanine transaminase (ALT) levels were also elevated in HFHSD WT, consistent with the findings of fatty liver. (Figure 1G). Serum triglycerides and total cholesterol were also lower in SOD1 −/− (Supplementary Figure S1). These results suggest that SOD1 −/− had less lipid storage than WT, even when fed on HFHSD, which may have contributed to the lack of weight gain.

Blood Glucose and Insulin Levels
Since lipid storage changes were observed in various organs, blood glucose and serum insulin levels were measured to determine insulin resistance. The OGTT (oral glucose tolerance test) showed similarly elevated blood glucose levels in both types of mice with HSHFD compared to ND (Figure 2A). On the other hand, serum insulin levels were lower in SOD1 −/− fed on HSHFD ( Figure 2B). Serum insulin divided by blood glucose level (HOMA-β equivalent), indicative of insulin secretion, was significantly smaller in SOD1 −/− given HFHSD ( Figure 2C). The product of blood glucose and serum insulin levels (HOMA-IR equivalent) indicates insulin resistance, and this index was elevated in WT given HFHSD; however, it was lower in SOD1 −/− given HFHSD ( Figure 2D). In WT, HFHSD gained weight and increased insulin resistance, which increased blood glucose, whereas, in SOD1 −/− , blood glucose was thought to increase due to decreased insulin secretion. The increase in blood glucose of SOD1 −/− fed on HFHSD was mild relative to decreased insulin.

Weight of Pancreatic β-Cells
Since insulin secretion was low in SOD1 −/− , we examined morphological changes in the islets of the pancreas. The ratio of β-cells to islets was measured by immunofluorescence staining for insulin and glucagon ( Figure 3A), and the weight of β-cells was estimated from the weight of the whole pancreas. WT on HFHSD increased pancreatic β-cells weight, but SOD1 −/− did not show any increase even on the HFHSD diet ( Figure 3B). This result is consistent with the fact that serum insulin was not elevated in SOD1 −/− on HFHSD.

Oxygen Consumption and Intake of the Diet
Next, oxygen consumption was measured to examine the effects of insulin. Insulin is well known to induce to transfer and express GLUT4 to the cell membrane by attaching to the insulin receptor. Glucose transferred through GLUT4 is metabolized to Acetyl-CoA, NADH 2 + , and FADH 2 is produced, and oxygen is consumed to produce ATP [21]. When insulin acts efficiently, oxygen consumption is thought to increase [22] and oxygen consumption is known to be depressed in insulin resistance [23][24][25]. Oxygen consumption was increased in SOD1 −/− fed on HFHSD compared to ND, whereas in WT, oxygen consumption was somewhat decreased in HFHSD compared to ND. ( Figure 4A). The food intake was increased despite smaller body weight in SOD1 −/− with HFHSD ( Figure 4B). Thyroid-stimulating hormone (TSH) and free thyroxine (free T4) levels did not differ among the four groups. Rectal temperature tended to be higher in the SOD1 −/− group, but there were no significant differences (Supplementary Figure S2). SOD1 −/− fed on HFHSD consumed more oxygen and food than WT, despite their smaller body weight. Insulin secretion was decreased in SOD1 −/− , but the effect of insulin action was thought to be increased.

ATP Levels and AMPK Expression in Skeletal Muscle
Mitochondria consume oxygen and produce ATP via oxidative phosphorylation and electron transport chain. Since SOD1 −/− fed on HFHSD consumed a lot of oxygen and diets, we hypothesized that there would be a lot of ATP in skeletal muscle. However, skeletal muscle ATP levels were lower in SOD1 −/− and significantly lower in the HFHSD compared to WT. ADP/ATP ratio was higher in SOD1 −/− with HFHSD ( Figure 5A,B).
AMPK (AMP-activated protein kinase) monitors intracellular energy status and regulates glucose and lipid metabolism to maintain and restore energy balance. AMPK is activated by phosphorylation of Thr172, and the increase in AMP/ATP ratio and ADP/ATP ratio associated with the decrease in ATP inhibits the dephosphorylation of Thr172, leading to its activation. Then, we checked the phosphorylation of Thr172 of AMPK by Western blotting. Consistent with the decrease in ATP, phosphorylation of AMPK was observed in skeletal muscles of SOD1 −/− with HFHSD ( Figure 5C). AMPK phosphorylation was also observed in adipocytes (data not shown) and liver ( Figure 5D) in the HFHSD group of SOD1 −/− .

ATP Levels and AMPK Expression in Skeletal Muscle
Mitochondria consume oxygen and produce ATP via oxidative phosphorylation and electron transport chain. Since SOD1 −/− fed on HFHSD consumed a lot of oxygen and diets, we hypothesized that there would be a lot of ATP in skeletal muscle. However,

Discussion
This study examined the role of O 2 · − in metabolic syndrome in vivo. WT and SOD1 −/− were fed on HFHSD to develop metabolic syndrome [14,15] and compared with ND. SOD1 is present in the cytoplasmic and mitochondrial intermembrane space and degrades O 2 · − , which may alter the dynamics of O 2 · − at that local site. Metabolic syndrome was induced by feeding on HFHSD [14,15] for 24 weeks, and as in previous reports [26], skeletal muscle from HFHSD-fed mice showed red fluorescence in DHE staining and even stronger fluorescence in SOD1 −/− , reflecting cytosolic O 2 · − . SOD1 −/− had reduced weight gain and less fat deposition in HFHSD. WT showed insulin resistance and elevated blood glucose in HFHSD, while SOD1 −/− showed decreased insulin secretion and elevated blood glucose. However, the increase in blood glucose was mild compared to the decrease in insulin, suggesting that insulin was more effective in HFHSD-fed SOD1 −/− . In WT, oxygen consumption was decreased in the HFHSD group compared to the ND group but instead increased in SOD1 −/− . On the other hand, skeletal muscle ATP levels were lower in SOD1 −/− fed on HFHSD, with concomitant phosphorylation of AMPK Thr172. These results suggest that catabolism is enhanced in SOD1 −/− fed on HFHSD.

Body Weight, Blood Glucose and Insulin Changes
SOD1 −/− had less body weight than WT, even in ND; induction of metabolic syndrome in HFHSD resulted in attenuated weight gain, similar to WT in ND. From both clinical data and animal models, it is well known that insulin promotes anabolism and contributes to weight gain [27,28]. Fasting insulin concentrations tended to be lower in SOD1 −/− with HFHSD than ND, and insulin secretion in response to blood glucose was significantly reduced, contributing to the lack of weight gain and adipose mass. It has also been reported that SOD1 −/− alters the gastrointestinal microbiota [29] and may impair absorption. Furthermore, hydrogen peroxide is produced by SOD1 from the O 2 · − and hydrogen peroxide is involved in cell hypertrophy [12]. SOD1 −/− was also claimed to be a model of sarcopenia showing various signs of aging [30]. These combined factors were thought to be responsible for the weight loss.
Blood glucose was elevated in SOD1 −/− with HFHSD but not high enough to account for the decrease in serum insulin levels. One mechanism for getting blood glucose into cells is GLUT4. Insulin activates AKT via insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3K), and AKT promotes GLUT4 translocation to the cell membrane [31,32]. Another regulator of GLUT4 is AMPK, which controls GLUT4 transport in an insulin-independent manner [33]. The reason why blood glucose was not higher in SOD1 −/− with HFHSD in the present study despite insulin levels may be that GLUT4 was upregulated by AMPK activation and the action of insulin was enhanced.
In WT with HFHSD, the estimated pancreatic β-cell weight increased, reflecting elevated serum insulin levels. In SOD1 −/− with HFHSD, insulin secretion was reduced; however, the estimated weight of β-cells remained the same and no morphological changes were observed. It was thought that the function of insulin secretion was impaired. Some studies showed that ROSs impaired insulin secretion [34,35]. On the other hand, there are reports that ROSs are involved in the mechanism of insulin resistance, and the mechanism was reported that chronic increase in ROSs led to phosphorylation of IRS-1 and decreased translocation of GLUT4 to the membrane [36]. In the present study, insulin resistance increased with increasing O 2 · − in WT with HFHSD, while in SOD1 −/− with HFHSD, insulin secretion decreased and insulin resistance instead improved. This discrepancy may be due to the type of ROSs and their localization. SOD removes one of the ROSs (O 2 · − ); however, at the same time, it produces another ROSs (hydrogen peroxide). The effects of ROSs are challenging to discuss in general, and the type of ROSs and their localization must be considered. Teratani et al. used SOD1 −/− and found that cytoplasmic hydrogen peroxide produced by SOD1 enhanced the amount and downstream signaling of DNA-bound PPARγ, leading to triglyceride accumulation in hepatocytes and liver [37].

Oxygen Consumption and AMPK Activation
It has been reported that mice on a high-fat diet consume less oxygen throughout the day than mice on a low-fat diet or a normal diet [23,24], and that fasting mice on the third day after starting a high-fat diet consume less oxygen than mice on a normal diet [25]. In this study, oxygen consumption was significantly reduced throughout the day in WTs with HFHSD. On the other hand, SOD1 −/− conversely increased oxygen consumption in HFHSD significantly. Dietary intake was significantly increased in SOD1 −/− with HFHSD, and body temperature tended to be higher in SOD1 −/− , although there were no significant differences. The prominent place where oxygen is consumed is in Complex IV within the mitochondrial electron transfer system, where the electrons used to form the proton gradient to generate ATP combines with oxygen to form water. Contrary to expectations, however, ATP concentrations were relatively low in the skeletal muscle of SOD1 −/− . Correspondingly, phosphorylated AMPK expression was elevated in SOD1 −/− . Regarding the relationship between AMPK and ROSs, while some reports suggest that exogenous hydrogen peroxide activates AMPK [38], others suggest it conversely inhibits its activation [39], and no sure view has been obtained. Hinchy et al. used O 2 · − producing MitoParaquat in the mitochondrial matrix and reported that ROS from mitochondria does not directly activate cytosolic AMPK, but mitochondrial ROS reduces the ATP/ADP ratio and then activates AMPK [13]. Mitochondria are one of the sites of ROS production, and it has been reported that O 2 · − is produced by electron leakage, which occurs at Complex I (reverse electron transport) [40] and Complex III [41]. O 2 · − generated in Complex I are released into the mitochondrial matrix, whereas O 2 · − generated in Complex III is released into both sides of the intermembrane [42]. SOD1 is present in the mitochondrial intermembrane space, and it is likely that the concentration of O 2 · − in the mitochondrial intermembrane space is increased in SOD1 −/− . The presence of O 2 · − in the mitochondrial matrix and the mitochondrial intermembrane space may have reduced the ATP/ADP ratio, resulting in this result.
One of the possible mechanisms by which ATP is reduced in this study is the effect of uncoupling protein (UCP). UCP is a mitochondrial transporter protein that causes proton leakage across the mitochondrial inner membrane and functions to relax the proton gradient formed by the electron transfer system without producing ATP. The skeletal muscle used in this study is rich in UCP3. In addition to the insufficient scavenging capacity of O 2 · − in the mitochondrial intermembrane space in SOD1 −/− , HFHSD loading may have further increased O 2 · − . It has been reported that UCP is activated by superoxide anion s [43], and thus UCP3 in skeletal muscle may have been activated in SOD1 −/− fed on HFHSD. The increased oxygen consumption suggests that a proton gradient was formed by a large amount of oxygen in the electron transfer system; however, this gradient may have been attenuated by UCP3 without ATP production. Furthermore, it has been reported that UCP2 is present in pancreatic beta cells and that UCP2 is important for pancreatic beta cell dysfunction by inhibiting insulin secretion [44]. Another possible mechanism is that mitochondria-generated ROS may induce posttranslational modifications in ATP synthase, thereby modulating its activity [45].
In SOD1 −/− , O 2 · − in the mitochondrial intermembrane space were further increased by HFHSD loading, which may have activated UCP. In skeletal muscle, the decrease in ATP production due to UCP3 activation leads to increased oxygen consumption, and the increase in ADP/ATP leads to AMPK activation. Activated AMPK promotes cellular uptake of blood glucose via GLUT4 [46,47], and blood glucose levels did not increase significantly. In pancreatic beta cells, UCP2 prevents the production of ATP, which is necessary for insulin secretion, resulting in decreased insulin secretion and impaired anabolism, which may have led to decreased fat retention and weight loss.

Conclusions
O 2 · − in the mitochondrial intermembrane space is metabolized by SOD1, but when accumulated, they inhibit ATP production, reduce insulin secretion, and promote metabolism in the catabolic direction. Proper metabolism of O 2 · − in the mitochondrial intermembrane space is thought to lead to insulin resistance, which leads to the accumulation of visceral fat and the development of Mets.


s2fieldsofstudy: Biology Chemistry

text:
The cyclic octapeptide antibiotic argyrin B inhibits translation by trapping EF-G on the ribosome during translocation

Significance The increase in multidrug-resistant bacteria highlights the urgent need for compounds with novel target sites that can be developed as antibiotics. The argyrins represent a family of naturally produced octapeptides that display promising activity against Pseudomonas aeruginosa by inhibiting protein synthesis. Our structural and kinetic analyses reveal that argyrins inhibit protein synthesis by interacting with, and trapping, the translation elongation factor G (EF-G) on the ribosome, analogous to that reported previously for the unrelated antibiotic fusidic acid. However, the binding site of argyrin on EF-G is distinct from that of fusidic acid, indicating that intramolecular movements at the domain III/V interface of EF-G are also essential for facilitating late events in the translocation mechanism.

but now in presence of the test synergistic partner compound (Appendix SI, Fig. S1A-F). The partner compound (0.5 μL) was added at a fixed final concentration that corresponded to its previously established IC50. The IC50 of each compound alone was then compared with the IC50 measured in the presence of the partner compound (Appendix SI, Fig. S1G-H). Since the in vivo (4) and in vitro (5) synergy between virginiamycin M1 (VrgM1) and virginiamycin S1 (VrgS1) is welldocumented, this combination was used as positive control for synergy. In addition, we considered the use of the erythromycin (Ery) as a negative control since macrolides such as erythromycin have a different mechanism of action, bind to the ribosome (and not EF-G), and the binding site is located far from ArgB-EF-G (6). Moreover, Ery and ArgB display IC50 values within the same order of magnitude. Collectively, the results reveal that the combination of VrgM1 and S1 leads to a 6-fold increase in inhibitory activity compared to when these compounds are used separately, whereas no significant increase was observed for all other combinations of compounds tested (Appendix SI, Fig. S1H).
Ribosome purification for co-sedimentation assays and the EF-G-ArgB-70S cryo-EM studies. Ribosomes were purified as previously described (7). In short, E. coli BL21 (2 L in LB media) was incubated at 37°C and harvested 5,000 x g and 4°C for 25 min in mid exponential phase. Cell pellets were resuspended in 20 mL of buffer A (25 mM HEPES-KOH, pH 7.4, 20 mM Mg(OAc)2, 100 mM KOAc, 1 mM DTT) and disrupted utilising a microfluidizer. The crude cell lysate was centrifuged at 21,000 x g for 20 min at 4 °C. Subsequently, the cleared lysate was layered on a high salt (800 mM KCl) 25% sucrose cushion in buffer A and centrifuged for 17.5 h at 39,000 rpm and 4°C in a Ti45 rotor (Beckmann coulter). The obtained ribosome pellet was resuspended in buffer A. 120 OD260 of the purified ribosomes were layered onto six 10-40% sucrose density gradients in buffer A and centrifuged for 3.5 h at 30,000 rpm and 4°C in a SW32 rotor (Beckmann coulter). Ribosome profiles were recorded at a wavelength of 254 nm using a gradient station (Biocomp). The 70S peaks were collected and subsequently pelleted for 14.5 h at 43,000 rpm and 4°C using a Ti45 rotor. The obtained pellets were resuspended in buffer A and following A260 concentration measurements was stored at -80°C.
Co-sedimentation assays. Co-sedimentation assays were performed as previously described (8). In short, 0.4 μM of purified E. coli 70S were mixed in buffer A (25 mM HEPES-KOH, pH 7.4, 20 mM Mg(OAc)2, 100 mM KOAc, 1 mM DTT) with 1 μM EF-G, 500 µM GTP and 50 μM of either argyrin B or fusidic acid. Reactions were incubated for 15 min at 37 °C and subsequently loaded on a 10% (w/v) sucrose cushion in buffer A and centrifuged for 35 min at 70,000 rpm in a TLA100 rotor at 4 °C. The pre-centrifugation reaction (R) and supernatant (S), as well as 10 pmol of the pellet (P), were applied to an 18% SDS-PAGE and stained with instant blue (Expedeon).
Data collection was performed using a FEI Titan Krios transmission electron microscope equipped with a Falcon II direct electron detector at 300 kV with a dose of 2.5 e -/Å 2 per frame, using a pixel size of 1.084 Å and a defocus range of -0.5 to -3.5 μm, resulting in 7,891 micrographs. The first ten frames (28 e -/Å 2 in total) were aligned using MotionCor2 (9). Power spectra, defocus values, astigmatism and estimation of micrograph resolution were determined by CTFFIND4 software (10). A resolution cut-off of 3.5 Å and defocus cut-off of 30,000 were applied. Following further visual inspection of micrographs for thon-rings and ice quality, 3,466 micrographs were used for automated particle picking by GAUTOMATCH (http://www.mrc-lmb.cam.ac.uk/kzhang). 610,946 particles were extracted using RELION 3.0 (11) and subjected to 2D-classification using 130 classes. 557,434 particles were focus-sorted with a soft-mask around EF-G into four classes for 300 rounds. Class II to IV contained density for EF-G, whereas class I appeared to contain a vacant 70S ribosome and was not further pursued. Contrast transfer function (CTF) and beam tilt refinement with subsequent Bayesian polishing was performed on class II to IV. Using the gold standard criterion (Fourier shell correlation of 0.143), post-processed maps showed an improved resolution by roughly 0.5 Å, resulting in average resolution of 3 Å for class II to IV. Class IV was subjected multi-body refinement with three masks, consisting of large subunit with EF-G, small subunit body and platform, and small subunit head. Resolutions were improved to 2.76 Å, 2.77 Å and 3 Å, respectively, reaching 2.6 Å in the large subunit core. The refined maps were sharpened by applying an automatically determined B factor using RELION 3.0 (11). Local resolution and maps filtered according to local resolution were computed using SPHIRE 1.3 (12). For detailed information on data collection parameters see Fig. S5 and Table S1.
Modelling for the EF-G-ArgB-70S complex. The INT2-FA-translocation intermediate structure (PBD-ID 7N2C), which was based on PDB-ID 4YBB (13), was used as a starting model (14), fit into the EM map and refined through iterative rounds of manual model building in Coot (v.0.9.4.1) (15,16), and real-space refinement using PHENIX (v.1.19-4092) (17,18). A 3D atomic model for Argyrin B was generated de novo from the chemical 2D structure (PubChem CID 9940787) using the Chemdraw-integrated Chem3D software. Restraints for Argyrin B were generated using PHENIX eLBOW (19). Molecular models were manually adjusted and refined using Coot (15,16). The atomic models were combined and refined using real space-refined with secondary structure restraints calculated by PHENIX (v1.14) (17,18). For detailed information on model-building statistics see Table S1. smFRET experiments. 70S ribosomes labelled with LD550 on uL1 and LD650 on uS13, programmed with 5′-biotinylated mRNA, containing P-site bound fMet-tRNA fMet and displaying the codon UUC in the A site were immobilized on passivated coverslips as described previously (21)(22)(23)(24). The ribosomes were then incubated for 2 minutes with EF-Tu ternary complex containing Phe-tRNA Phe , leading to stoichiometric formation of PRE ribosomes containing A-site fMet-Phe-tRNA Phe , P-site tRNA fMet . To initiate translocation, 5 µM EF-G with 1 mM GTP, with or without 10 µM ArgB or 10 µM FA, was delivered to the flow cell by stopped-flow injection. All smFRET experiments were carried out at 25°C and ribosomes, tRNA, EF-Tu and EF-Ts were prepared as previously described (23,24). The time-evolution of the FRET signal was then recorded using a home-built total internal reflection-based fluorescence microscope (25) at ~0.1 kW/cm 2 laser (532 nm) illumination at a time resolution of 40 or 400 ms. Donor and acceptor fluorescence intensities were extracted from the recorded movies and FRET efficiency traces were calculated using the SPARTAN software implemented in MatlabR2015b (25). FRET traces were selected for further analysis according to the following criteria: a single catastrophic photobleaching event, at least 8:1 signal/background-noise ratio and 6:1 signal/signal-noise ratio, less than four donor-fluorophore blinking events, a correlation coefficient between donor and acceptor <0.5. To accurately estimate the FRET efficiencies of the major states occupied by the ribosome during translocation histograms were constructed using the first 50 frames of all FRET efficiency traces for each condition. Three gaussian functions were then fit to each histogram to estimate the mean FRET intensity of the PRE, intermediate and POST (either EF-G-bound in the presence of the drugs or unbound in their absence) states.

Buffers and reagents for smFRET and EF-G-
Purification of reagents for EF-G-ArgB-INT2 cryo-EM structure determination. Wild-type ribosomal subunits were purified from E. coli MRE600 for cryo-EM experiments as previously described (20). EF-Tu/EF-Ts (22) and EF-G (21) were purified as previously described. E. coli tRNA fMet and tRNA Phe were purified as previously described (23). To prepare Phe-tRNA Phe ternary complex, phenylalanine (1 mM Cryo-EM grid preparation for EF-G-ArgB-INT2 structure. Cryo-EM grids were prepared using a Vitrobot Mark IV plunge-freezing device (Thermo Fisher Scientific). For each experiment, 3 μl of sample was applied to UltrAuFoil R 1.2/1.3 gold 300 mesh grids that had been glow-discharged (Ar/O2) for 20 s using a Solarus II Plasma Cleaning system (Gatan). Grids immediately blotted in the Vitrobot chamber (4°C, 100%, 4.5-5.5 s blot time; -5 blot force) and plunged into liquid ethane.

Data collection and single particle reconstruction for EF-G-ArgB-INT2 cryo-EM studies.
Cryo-EM data for the translocation intermediate structure were collected using a Titan Krios G3i (Thermo Fisher Scientific) transmission electron microscope equipped with a K3 direct electron detector and post column GIF (energy filter). K3 gain references were acquired just before data collection. Data collection was performed using an image shift protocol (nine images were collected with one defocus measurement per nine holes). Movies were recorded at defocus values from −0.5 μm to −2.0 μm at a magnification of 130,000×, which corresponds to the pixel size of 0.648 Å per pixel at the specimen level (super-resolution 0.324 Å per pixel). During the 2-s exposure, 70 frames (0.03 s per frame, dose/frame 1 e − per frame per Å 2 ) were collected with a total dose of around 70 e − per Å 2 . Motion correction was performed on raw super-resolution movie stacks and binned twofold using MotionCor2 software (9). CTF parameters were determined using CTFFind4.1 (10) and refined later in Relion 4.0 (26). All pre-processing and particle sorting were conducted in Relion 4.0 (26). Sharpened and locally filtered maps were used to aid in model building. Pixel size was calibrated against high resolution 70S crystal structure PDB-ID 4YBB. Cryo-EM density map values were normalized to mean = 0 and standard deviation (σ) = 1 in UCSF Chimera (27) using the vop scale function. For detailed information on data collection parameters see Fig. S5 and Table S1. The INT2-FA-translocation intermediate structure  (PDB-ID 7N2C), which was based on PDB-ID 4YBB (13), was used as a starting model (14), fit into the EM map and refined through iterative rounds of manual model building in Coot (v.0.9.4.1) (15,16), and real-space refinement using PHENIX (v.1.19-4092) (17,18). mRNA nucleotide 37 corresponds to the +1 position. Restraints for refinement were generated using phenix.elbow (19). Models were validated using phenix.validation_cryoem (28) with the built-in MolProbity (29) scoring. See Table S1 for more information.       (26). Particles from two datasets were refined separately for initial reconstructions. Subsequently, a focus ligand mask containing EF-G, P-site tRNA, and E-site tRNA was applied for sorting each reconstruction into ten classes. Class X of dataset 1 and Class VIII of dataset 2 contained EF-G. Classes containing EF-G were combined, refined, and subjected to another round of classification with a ligand mask into five classes. The EF-G-containing class was refined and subjected to a final round of unmasked classification into eight classes. Class II containing two tRNAs and EF-G was refined (undecimated) followed by CTF-refinement, Bayesian polishing, and Post Processing.       Global alignment (top) and of EF-G from the EF-G-ArgB-INT2 complex (coloured by domain) to S. cerevisiae eEF2 (grey) bound to Sordarin (yellow) and zoom-in of drug binding site between domains III and V of EF-G/eEF2.