Microtubule disassembly promotes ERM activation. (A) Distribution of the 3,469 compounds (blue dots) screened over MyrPB-ezrin-rLucII BRET2 activation. BRET2 signals are normalized to vehicle (DMSO, 100%). Grey zone represents vehicle (100%) ± 28.4% corresponding to three times the SD of all compounds tested. Compounds promoting a BRET2 signal decrease lower than 71.6% are potential ezrin activators. (B) Schematic representation of the number of hits identified after both the primary and confirmation screens. (C) Table showing the targets of each compounds identified and validated in the chemical screen. BRET2 signals (in % compared to vehicle) measured for each compound in the primary and confirmation screens are indicated in columns 2 and 3, respectively. (D) Immunofluorescence of microtubules of HEK293T cells treated with indicated inhibitors for 15 min. (E and F) Immunoblots of HEK293T (left) and HeLa (right) cells incubated with the indicated concentrations of podophyllotoxin for 15 min (E). p-ERM over ERM signals were quantified and normalized to vehicle (DMSO; F). (G) Immunoblots of Hs578T, A375, SW620, and MC38 cells treated with 1 µM nocodazole for 15 min. (H and I) Immunofluorescence of HEK293T cells incubated with vehicle (DMSO) or 1 µM podophyllotoxin for 15 min (H). p-ERM staining at the plasma membrane was quantified and normalized to cells treated with vehicle (I; n > 45 cells). Immunofluorescence (H) and quantification of p-ERM staining at the plasma membrane (I) of vehicle treated cells were already shown in Fig. 1, H and I, respectively. (J) Immunoblot of HEK293T cells transfected with ezrinWT-rLucII or ezrinKK211,212MM-rLucII and treated with vehicle (DMSO) or 1 µM nocodazole for 15 min. Immunofluorescences (D and H) and immunoblots (E, G, and J) are representative of at least two independent experiments. p-ERM quantifications represent the mean ± SD of at least two independent experiments. Dots represent independent experiments (F) or individual cells (I). P values were calculated using one-sample t test (F) or using two-tailed unpaired t test (I). *, P < 0.05; ****, P < 0.0001. Numbers associated with Western blots indicate molecular weight in kD. Source data are available for this figure: SourceData FS1.