lower quartile=15.4/15.2/15.4/14.9/14.7/15, minimum=10.9/10.7/11.1/9.13/9.75/10.3. Normalized intensities Quant1/ Quant2/ Quant3/ Quant4/ Quant5/ Quant6: n=5228/5228/5228/ 5228, maximum=22.6/22.6/22.6/22.7/22.6/22.6, upper quartile=15.9/15.9/15.9/15.9/15.9/15.9, lower quartile=15. 

GTCTTGCCATGCCACTTGTC Hu

-MUT-F TGGGTTTGCCAACTGTGAAAA Hu-MUT-R GGTATTCCCTCAGCTACAGCTT Hu-PCCA-F AAGCTACCTCAACATGGATGC Hu-PCCA-R GTGTCAGGTCCAATGAAAACGA Hu-MCEE-F ATGTAGCCATAGCAGTGCCAG Hu-MCEE-R GACTGTCACGTCCCAATGGAT Hu-ALDH6A1-F GGCAGACACTTCAGTATTAAGCC Hu-ALDH6A1-R AGAGGCAGACGGTAGGAATAAA Hu-ACADM-F ACAGGGGTTCAGACTGCTATT Hu-ACADM-R TCCTCCGTTGGTTATCCACAT Hu-HSD17B10-F TGGCGGTAATAACCGGAGGA Hu-HSD17B10-R ACAGTTGACAGCTACATCCACA Hu-ACAA1-F GCGGTTCTCAAGGACGTGAAT Hu-ACAA1-R GTCTCCGGGATGTCACTCAGA Hu-ACAA2-F CTGCTCCGAGGTGTGTTTGTA Hu-ACAA2-R GGCAGCAAATTCAGACAAGTCA Hu-PCCB-F TCTGCAAAATCATGGACCAGG Hu-PCCB-R GGATGCCGTAACATTCCTCAG Hu-MCCC1-F TGATGATGCTATGCTGATCGAGA Hu-MCCC1-R GATGTCGCCTCTGCACACTA Hu-MCCC2-F GAGGTGCCTACTACCCAGTGA Hu-MCCC2-R GCTCCTCCCGAATCAACTAAGT Hu-AUH-F ATTGGCGATTATTCCTGGTGG Hu-AUH-R GAACGTGGCTGATTAAGCCCA Hu-HMGCL-F TCCACTGCCATGACACCTATG Hu-HMGCL-R AAGCCCTCTAGCATGTAGACC Hu-HMGCS-F CATTAGACCGCTGCTATTCTGTC Hu-HMGCS-R TTCAGCAACATCCGAGCTAGA Hu-IVD-F ATGGCAGAGATGGCGACTG Hu-IVD-R TAGCCCATTGATTGCATCGTC mouse-MCCC1-F TGCACACCCATCTTTTTGGCT mouse-MCCC1-R ACCATGAAGTATGGAACAACCC mouse-ACAA1-F CGCTCAGAAATTGGGCGATG mouse-ACAA1-R TCTCCAGGACGTGAGGCTAAA mouse-AUH-F AGTCAGGAGTCACTTCTGGCT mouse-AUH-R AGCTGGCTCTAGCGTGTGA mouse-ACAA2-F AGCTCTGCATGACATTGCCC mouse-ACAA2-R CTGCTACGAGGTGTGTTCATC mouse-DLD-F CCTATCACTGTCACGTCAGCC mouse-DLD-R GAGCTGGAGTCGTGTGTACC mouse-HIBADH-F ACACGTCATAGAGGATGAGTGG mouse-HIBADH-R GCAGCGGTGTGTTCTAGGTC mouse-BCKDHA-F CATTGGGCTGGATGAACTCAA mouse-BCKDHA-R CTCCTGTTGGGACGATCTGG mouse-IVD-F CTCCTCGTTTAGCCCGTTGA mouse-IVD-R GGACGGCGAGTTTCCAGTT mouse-MCEE-F TTTTCCAAATCTGGCACTGCTA mouse-MCEE-R ATGAGGCGTGTAGTGAAGGC mouse-ALDH6A1-F TCCCTTGTTCCAGTGTGATTAAC mouse-ALDH6A1-R AGCCGTTGAGTCCTGCAAAC mouse-HMGCS2-F GTCCACATATTGGGCTGGAAA mouse-HMGCS2-R GAAGAGAGCGATGCAGGAAAC mouse-ALDH1B1-F CATGCCACTCGTTGTTGATGA mouse-ALDH1B1-R CTCCAGGGCAGGACTACCTC mouse-PCCB-F TGCGCTCTTTAACTGAAACCG mouse-PCCB-R GCGGCGATTAGGATTCGGG mouse-HSD17B10-F TGGGGCAAATATGCAGCTTTC mouse-HSD17B10-R GCTTGGTCGCGGTAGTAACTG mouse-ALDH9A1-F AGGCCACTTTTCTTACTCCAGA mouse-ALDH9A1-R GGCCGAGTGATTGCCACTT mouse-MUT-F CCTCTGGGTTTTTGCCTTTCAG mouse-MUT-R

a

  1/15.1/15.1/15.1/15.1/15.1, minimum=10.6/10.6/10.8/9.35/10.1/10.4. b The heatmap of the differential proteins from Huh7 cells with or without glucose deprivation treatment. c PROX1 peptides identified through liquid chromatography-tandem mass spectrometry are shown. d, eWestern blot analysis the level of PROX1 in the Huh7 cells(d) and MEFs (e) in response to glucose starvation (Glc starv.) as indicated. f Immunofluorescence assay shows the expression of PROX1 in the MEFs upon glucose starvation and metformin treatment (5mM) for 12h. Scale bar, 10 µm. The immunofluorescences are repeated independently with similar results at three times. g Western blot analysis the level of PROX1 in the Huh7 cells in response to MK8722, a selective on-target synthetic AMPK activator. h Liver tissues from normal, fasted and metformin (500mg/L) treatment mice for 24h are subjected to western blot (n=4). i The western blot analysis of the Huh7 and HepG2 cells were infected with the lentivirus either expressing PROX1 siRNA (si259 or si1646) precursor or scrambled siRNA precursor (SCR). j Gating strategy to determine the percentage of apoptotic cells. k The apoptotic analysis of the Huh7 cells were infected with the lentivirus as the indicated by flow cytometry. l The western blot analysis of the wild-type (WT) and AMPKα knockout (Ampkα -/-) MEFs were infected with the lentivirus either expressing scrambled siRNA precursor (SCR) or Prox1 siRNA (shProx1) as the indicated. m Immunohistochemistry analysis of the levels of Lkb1 and Ampkα in the murine lung tumour tissues from Kras G12D , Kras G12D ; Lkb1 flox/flox (Kras G12D /Lkb1 L/L ) and Ampkα KO Kras G12D (Kras G12D -sgAmpk) mouse (n=5). Scale bar, 50 µm. n The western blot analysis of the cell lysates as indicated. The immunoblots are repeated independently with similar results at three times. Source data are provided as a Source data file. Supplementary Fig. 2 PROX1 is a novel substrate of AMPK. a, b Realtime-PCR assay shows the expression of PROX1 in the Huh7 and HepG2 cells upon glucose starvation (a) and metformin treatment (b) as indicated (n=3 independent experiments). c-e Huh7 and HepG2 cells upon glucose starvation or not were subject to cycloheximide (CHX, 100mg/mL) experiment as indicated. Representative western blot (c) and the corresponding quantified graph of Huh7 (d) and HepG2 (e) cells are shown. n=3 biologically independent experiments. f HEK293T transfected with FLAG-PROX1 or control plasmid were subject to Co-IP/MS. The assay is repeated independently with similar results at two times. g HEK293T cells expressing FLAG-PROX1 fragment incubated with purified GST-AMPKα2. The interaction was detected by anti-FLAG mAb after GST-pulldown as indicted. h Mapping of the PROX1interacting regions of AMPKα2. Upper, the domain organization of AMPKα2 and the deletion constructs. KD, kinase domain; RD, regulatory domain; Lower, co-IP was performed with an anti-FLAG mAb and captured PROX1 was detected by an anti-PROX1 polyclonal antibody as indicated. i Each GST-PROX1 fragment (left), purified recombinant PROX1 fragment (P1-WT) and replacement of S79 with Ala (A) in the PROX1 (P1-S79A) incubated with or without purified AMPK. p-AMPK substrate was detected by anti-phospho-AMPK substrate motif mAb after GST-pulldown. j Specificity of phosphor-S79-PROX1 antibody was determined by dot blot assay (upper). PVDF membrane was spotted with indicated amounts of phosphor-S79 peptide or un-phosphorylated peptide, probed with pS79-PROX1 antibody. Immunoblot analysis of the cell lysates from HEK293T cells expressing FLAG-PROX1 variants with the indicated antibodies (down). k Immunofluorescence analysis of the Huh7 cells expressing EGFP-PROX1 variants with the indicated antibodies (n=3 independent experiments). Scale bar, 25 µm. IB, immunoblot; IP, immunoprecipitation. Relative ratio of individual protein abundance was determined from signal intensity. The immunoblots are repeated independently with similar results at least three times. For a, b, d and e, data represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student's t-test. Source data are provided as a Source Data file. Supplementary Fig. 3 PROX1 is a novel substrate of CUL4-DDB1 E3 ligase complex, which directly reduces PROX1 protein stability. a Co-IP/MS was conducted to identify E3 ligase associated with PROX1 upon glucose starvation and MG132 treatment (4uM) for 8h. b The peptides of CUL4A (left), CUL4B (middle), and DDB1 (right) identified by Co-IP/MS are shown as the indicated. c Endogenous PROX1 in the Huh7 cells treated with MG132 (4uM) was immunoprecipitated using anti-PROX1 antibody or isotype IgG control and subjected to immunoblot analysis. d-f HEK293T cells were co-transfected with FLAG-PROX1 fragment and HA-CUL4A (d), and HA-CUL4B (e), or DDB1 (f) plasmids. Immunoblot analysis of the cell lysates and FLAG-IP with the indicated antibodies. g Immunoblot analysis of the cell lysates from HepG2 cells with the indicated antibodies. h, i Huh7 cells infected with Control and CUL4A lentivirus were subject to cycloheximide (CHX, 100mg/mL) experiment as indicated. n=3 biologically independent experiments. Representative western blot (h) and the corresponding quantified graph (i) are shown. j, k Huh7 cells infected with Control and DDB1 lentivirus were subject to cycloheximide (CHX, 100mg/mL) experiment as indicated. n=3 biologically independent experiments. Representative western blot (j) and the corresponding quantified graph (k) are shown. l Ubiquitination levels of PROX1 following knockdown of CUL4A, 4B and DDB1 as the indicated. m Ubiquitination levels of PROX1 in the Huh7 cells treated with MG132 (4uM) with or without glucose starvation were immunoprecipitated using anti-PROX1 antibody or isotype IgG control and subjected to immunoblot analysis. IB, immunoblot; IP, immunoprecipitation. The immunoblots are repeated independently with similar results at least three times. For i and k, data represent the mean ± SD. Statistical significance was assessed using twotailed unpaired Student's t-test. Source data are provided as a Source Data file. Heatmap demonstration of the gene expression related to valine, leucine and isoleucine degradation (BCAA metabolism) in the RNA-seq data from Huh7 cells infected with the lentivirus expressing PROX1 siRNA (si1646) or scrambled siRNA precursor (SCR). b GSEA shows the enrichment of BCAA metabolism in the PROX1 knockdown Huh7 cells. Statistical significance was assessed using Permutation test. c, dRealtime-PCR analysis the relative mRNA levels of BCAA metabolism genes from Huh7 (c) and HepG2 (d) cells infected with the lentivirus either expressing PROX1 siRNA (si259 or si1646) precursor or scrambled siRNA precursor (SCR) as indicated (n=3 independent experiments). e Realtime-PCR analysis the relative mRNA levels of BCAA metabolism genes from Prox1 f/f (WT) and Alb-Cre; Prox1 f/f (Prox1-cKO) mice treated as indicated (n=3). f ChIP-qrtPCR was performed with sonicated chromatins immunoprecipitated from Huh7 cells by anti-PROX1 mAb or pre immune IgG (control) (n=3 independent experiments). g KEGG pathway enrichment analysis the ATAC-seq results from WT and Prox1-cKO mice using the differentially opened at the promoters of genes (log2Fold change>1 or <-1 and padj<0.001). Statistical significance was assessed using hypergeometric test. h Density maps for ATAC-seq at BCAA metabolism genes from WT and Prox1-cKO mice. i Heatmap of the gene peaks by Chip-seq from Huh7 cells as indicated with H3K27ac antibody. j Density maps for H3K27ac at BCAA metabolism genes. For c, d, e and f, data represent the mean ± SD and *P<0.05, **P<0.01, ***P<0.001. Statistical significance was assessed using twotailed unpaired Student's t-test. Source data are provided as a Source Data file. 


Supplementary Information


AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis

Yanan Wang, Mengjun Luo, Fan Wang, Yu Tong, Linfeng Li, Yu Shu, Ke Qiao, Lei Zhang, Guoquan Yan, Jing Liu, Hongbin Ji, Youhua Xie, Yonglong Zhang, Wei-Qiang Gao, Yanfeng Liu Supplementary Fig. 1