Figure 2A . SIRT7 binds to PRMT1 , PRMT3 , and PRMT6 . GFP - tagged PRMTs ( PRMT1 - 9 ) were expressed in HEK293T cells . PRMT protein was immunoprecipitated with GFP antibody and analyzed by western blotB . Co - expression of PRMT6 , but not PRMT1 or PRMT3 , increases R388 methylation of SIRT7 . Flag - tagged SIRT7 and GFP - tagged PRMT1 , PRMT3 , or PRMT6 were co - expressed in HEK293T cells . R388 methylation of immunopurified SIRT7 was analyzed by western blotC . Wildtype PRMT6 , but not its catalytic - deficient mutant , upregulates SIRT7 R388 methylation . Wildtype PRMT6 or its V86K / D88A mutant ( DM ) was co - expressed with Flag - tagged SIRT7 in HEK293T cells . R388 methylation of immunopurified SIRT7 was detected by western blotD - E . PRMT6 physically interacts with endogenous SIRT7 . Endogenous SIRT7 ( D ) or PRMT6 ( E ) was pulled down using corresponding antibody from HEK293T , MEF , and primary mouse hepatocytes , respectively . Input and immunoprecipitates were analyzed by western blotF . SIRT7 associates with PRMT6 in vitro . Recombinant GST - PRMT6 were incubated with HEK293T lysates . After GST pulldown , the interaction of recombinant protein with endogenous SIRT7 was analyzed by western blot . The arrows indicate GST and GST - PRMT6 protein , respectivelyG . PRMT6 inhibitor reduces arginine methylation of wildtype SIRT7 , but not RK / RF mutants . Wildtype SIRT7 or its RK / RF mutants were stably expressed in MEF cells . After treating cells with or without PRMT6 - specific chemical inhibitor ( PRMT6i ) for 24 hrs , SIRT7 was immunoprecipitated and blotted with R388 - specific methylation antibodyH . Prmt6 depletion decreases R388 methylation of endogenous Sirt7 . Scramble control cells ( scr ) or MEF cells stably expressing two different shRNAs ( # 1 and # 2 ) against Prmt6 was established . R388 methylation of immunopurified endogenous Sirt7 was determined by western blotI . Treatment with PRMT6 inhibitor reduces R388 methylation of endogenous SIRT7 in a dose - dependent manner . MEF and HEK293T cells were treated with increasing doses of PRMT6i for 24 hrs as indicated . R388 methylation of immunoprecipitated endogenous SIRT7 was determined by western blotJ - K . Prmt6 depletion reduces the percentage of R388 - methylated endogenous SIRT7 in MEF cells . R388 - methylated endogenous SIRT7 was captured from scramble control cells ( scr ) and Prmt6 - knockdown MEF cells by using R388 site - specific methylation antibody ( J ) . Input , output , and immunoprecipitate were analyzed by western blot to quantify the percentage of R388 - methylated protein ( K ) ( mean ± SD , n = 3 experimental replicates ) L . Recombinant PRMT6 directly methylates SIRT7 at R388 in vitro . Bacterially expressed PRMT6 and SIRT7 proteins were incubated with SAM or PRMT6i as indicated . Heat - inactivated PRMT6 was included as negative control R388 methylation of recombinant SIRT7 protein was determined by western

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. SIRT7 binds to PRMT1, PRMT3, and PRMT6. GFP-tagged PRMTs (PRMT1-9) were expressed in HEK293T cells. PRMT protein was immunoprecipitated with GFP antibody and analyzed by western blotB. Co-expression of PRMT6, but not PRMT1 or PRMT3, increases R388 methylation of SIRT7. Flag-tagged SIRT7 and GFP-tagged PRMT1, PRMT3, or PRMT6 were co-expressed in HEK293T cells. R388 methylation of immunopurified SIRT7 was analyzed by western blotC. Wildtype PRMT6, but not its catalytic-deficient mutant, upregulates SIRT7 R388 methylation. Wildtype PRMT6 or its V86K/D88A mutant (DM) was co-expressed with Flag-tagged SIRT7 in HEK293T cells. R388 methylation of immunopurified SIRT7 was detected by western blotD-E. PRMT6 physically interacts with endogenous SIRT7. Endogenous SIRT7 (D) or PRMT6 (E) was pulled down using corresponding antibody from HEK293T, MEF, and primary mouse hepatocytes, respectively. Input and immunoprecipitates were analyzed by western blotF. SIRT7 associates with PRMT6 in vitro. Recombinant GST-PRMT6 were incubated with HEK293T lysates. After GST pulldown, the interaction of recombinant protein with endogenous SIRT7 was analyzed by western blot. The arrows indicate GST and GST-PRMT6 protein, respectivelyG. PRMT6 inhibitor reduces arginine methylation of wildtype SIRT7, but not RK/RF mutants. Wildtype SIRT7 or its RK/RF mutants were stably expressed in MEF cells. After treating cells with or without PRMT6-specific chemical inhibitor (PRMT6i) for 24 hrs, SIRT7 was immunoprecipitated and blotted with R388-specific methylation antibodyH. Prmt6 depletion decreases R388 methylation of endogenous Sirt7. Scramble control cells (scr) or MEF cells stably expressing two different shRNAs (#1 and #2) against Prmt6 was established. R388 methylation of immunopurified endogenous Sirt7 was determined by western blotI. Treatment with PRMT6 inhibitor reduces R388 methylation of endogenous SIRT7 in a dose-dependent manner. MEF and HEK293T cells were treated with increasing doses of PRMT6i for 24 hrs as indicated. R388 methylation of immunoprecipitated endogenous SIRT7 was determined by western blotJ-K. Prmt6 depletion reduces the percentage of R388-methylated endogenous SIRT7 in MEF cells. R388-methylated endogenous SIRT7 was captured from scramble control cells (scr) and Prmt6-knockdown MEF cells by using R388 site-specific methylation antibody (J). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (K) (mean±SD, n=3 experimental replicates)L. Recombinant PRMT6 directly methylates SIRT7 at R388 in vitro. Bacterially expressed PRMT6 and SIRT7 proteins were incubated with SAM or PRMT6i as indicated. Heat-inactivated PRMT6 was included as negative control R388 methylation of recombinant SIRT7 protein was determined by western blot


Figure 3A . RF , but not RK mutant , suppresses H3K18 deacetylase activity of SIRT7 . Flag - SIRT7 and its RK / RF mutants were expressed in HEK293T and MEF cells , respectively . Cell lysates were subjected to western blot to detect histone H3K18 acetylation . H3K27ac and H3K56ac were included as negative control . The level of each histone acetylation marker was normalized against histone H3 ( Ratio ) B . R388 methylation of SIRT7 decreases its H3K18 deacetylase activity . HEK293T cells expressing wildtype SIRT7 or its RK / RF mutants were treated with or without AdOx for 24 hrs . The SIRT7 enzyme was immunopurified with Flag - beads and further incubated with extracted chromatin in vitro . R388 methylation of immunopurified SIRT7 and H3K18ac of in vitro deacetylated chromatin were determined by western blotC . RF , but not RK mutant , shows reduced H3K18 deacetylase activity in vitro . Chromatin was extracted from HEK293T cells and incubated with recombinant GST - tagged SIRT7 ( wildtype and RK / RF mutants ) in vitro for 2 hrs . H3K18ac was determined by western blotD . Wildtype PRMT6 , but not its methylase - dead mutant , decreases the H3K18 deacetylase activity of SIRT7 . Flag - tagged SIRT7 was co - expressed with wildtype PRMT6 or its catalytic - inactive mutant ( DM ) in HEK293T cells . The H3K18 deacetylase activity of immunopurified SIRT7 was assayed in vitroE . PRMT6 deficiency promotes the H3K18 deacetylase activity of SIRT7 . Control sgRNA or two independent sgRNAs ( # 1 and # 2 ) against PRMT6 was introduced into HEK293T cells . Flag - tagged SIRT7 was ectopically expressed in these cells . The histone deacetylase activity of immunoprecipitated SIRT7 was assayed in vitro using chromatin as a substrate . R388 methylation of immunopurified SIRT7 and H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blotF - G . Methylated recombinant SIRT7 shows reduced H3K18 deacetylase activity . In vitro methylated SIRT7 from Fig 2L was subjected to in vitro deacetylation assay . H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blot ( F ) . Relative H3K18ac level was quantified from three independent experiments ( G ) ( mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 , n . s . = not significant , unpaired two - tailed t test )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. RF, but not RK mutant, suppresses H3K18 deacetylase activity of SIRT7. Flag-SIRT7 and its RK/RF mutants were expressed in HEK293T and MEF cells, respectively. Cell lysates were subjected to western blot to detect histone H3K18 acetylation. H3K27ac and H3K56ac were included as negative control. The level of each histone acetylation marker was normalized against histone H3 (Ratio)B. R388 methylation of SIRT7 decreases its H3K18 deacetylase activity. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. The SIRT7 enzyme was immunopurified with Flag-beads and further incubated with extracted chromatin in vitro. R388 methylation of immunopurified SIRT7 and H3K18ac of in vitro deacetylated chromatin were determined by western blotC. RF, but not RK mutant, shows reduced H3K18 deacetylase activity in vitro. Chromatin was extracted from HEK293T cells and incubated with recombinant GST-tagged SIRT7 (wildtype and RK/RF mutants) in vitro for 2 hrs. H3K18ac was determined by western blotD. Wildtype PRMT6, but not its methylase-dead mutant, decreases the H3K18 deacetylase activity of SIRT7. Flag-tagged SIRT7 was co-expressed with wildtype PRMT6 or its catalytic-inactive mutant (DM) in HEK293T cells. The H3K18 deacetylase activity of immunopurified SIRT7 was assayed in vitroE. PRMT6 deficiency promotes the H3K18 deacetylase activity of SIRT7. Control sgRNA or two independent sgRNAs (#1 and #2) against PRMT6 was introduced into HEK293T cells. Flag-tagged SIRT7 was ectopically expressed in these cells. The histone deacetylase activity of immunoprecipitated SIRT7 was assayed in vitro using chromatin as a substrate. R388 methylation of immunopurified SIRT7 and H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blotF-G. Methylated recombinant SIRT7 shows reduced H3K18 deacetylase activity. In vitro methylated SIRT7 from Fig 2L was subjected to in vitro deacetylation assay. H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blot (F). Relative H3K18ac level was quantified from three independent experiments (G) (mean±SD, n=3 experimental replicates, **p<0.01, n.s.=not significant, unpaired two-tailed t test)


Figure 4Wildtype SIRT7 or its RF mutant was re - expressed in Sirt7 - knockdown MEF cells ( A ) Wildtype SIRT7 , but not its RF mutant , restores mitochondria mas in Sirt7 - deficient MEF cells Mitochondria mass was determined by Mito Tracker Red staining ( B ) ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant , unpaired two - tailed t test ) Wildtype SIRT7 , but not its RF mutant , restore ATP leve in Sirt7 - deficient MEF cells Cellular ATP was quantified and normalized to cell number ( C ) ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant , unpaired two - tailed t test ) Wildtype SIRT7 , but not its RF mutant , restore cellular respiration in Sirt7 - deficient MEF cells Respiration flux was determined as indicated ( D ) . ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant unpaired two - tailed t test ) Wildtype SIRT7 , but not its RF mutant , downregulates SIRT7 target genes involved in mitochondria biogenesis . Wildtype SIRT7 and its RF mutant were stably expressed in MEF cells ( E ) Wildtype SIRT7 , but not its RF mutant , downregulates SIRT7 target genes involved in mitochondria biogenesis SIRT7 target gene expression was determined and presented as a heatmap on a log2 scale ( F ) Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs . The H3K18ac level at the promoter of SIRT7 target genes ( Gfm2 , Mrps31 , Mrps33 , and Mrpl40 ) was determined by ChIP - qPCR , Rabbit IgG was used as a negative control ( G ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t testInhibition of Prmt6 decreases mitochondria mas of control cells , but not Sirt7 - knockdown MEF cells Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red staining ( H ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t testInhibition of Prmt6 decrease ATP leve of control cells , but not Sirt7 - knockdown MEF cells . Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Cellular ATP was quantified and normalized to cell number ( I ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t testInhibition of Prmt6 decrease the respiration flux of control cells , but not Sirt7 - knockdown MEF cells . Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated ( J ) . ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Wildtype SIRT7 or its RF mutant was re-expressed in Sirt7-knockdown MEF cells (A)Wildtype SIRT7, but not its RF mutant, restores mitochondria mas in Sirt7-deficient MEF cells Mitochondria mass was determined by Mito Tracker Red staining (B) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)Wildtype SIRT7, but not its RF mutant, restore ATP leve in Sirt7-deficient MEF cells Cellular ATP was quantified and normalized to cell number (C) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)Wildtype SIRT7, but not its RF mutant, restore cellular respiration in Sirt7-deficient MEF cells Respiration flux was determined as indicated (D). (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant unpaired two-tailed t test)Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis. Wildtype SIRT7 and its RF mutant were stably expressed in MEF cells (E)Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis SIRT7 target gene expression was determined and presented as a heatmap on a log2 scale (F)Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs. The H3K18ac level at the promoter of SIRT7 target genes (Gfm2, Mrps31, Mrps33, and Mrpl40) was determined by ChIP-qPCR, Rabbit IgG was used as a negative control (G) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t testInhibition of Prmt6 decreases mitochondria mas of control cells, but not Sirt7-knockdown MEF cells Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red staining (H) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t testInhibition of Prmt6 decrease ATP leve of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Cellular ATP was quantified and normalized to cell number (I) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t testInhibition of Prmt6 decrease the respiration flux of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated (J). ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test


Figure 5A . Glucose depletion reduces SIRT7 methylation . MEF cells stably expressing Flag - SIRT7 were cultured in medium depleted with glucose ( 12 hrs ) , FBS ( 12 hrs ) or glutamine ( 24 hrs ) . R388 methylation of immunopurified SIRT7 and Ampk phosphorylation were determined by western blot . R388 methylation level was normalized to Flag - SIRT7 ( Ratio ) B . Glucose starvation decreases R388 methylation of SIRT7 in a time - dependent manner . MEF cells stably expressing Flag - SIRT7 were cultured in glucose - free media as indicated . R388 methylation level of immunopurified SIRT7 was determined by western blotC . Glucose dose - dependently upregulates R388 methylation of SIRT7 . MEF cells stably expressing Flag - SIRT7 were cultured with increasing concentrations of glucose as indicated for 12 hrs . R388 methylation of SIRT7 and Ampk phosphorylation were determined by western blotD . AICAR treatment decreases R388 methylation of SIRT7 . MEF cells stably expressing Flag - SIRT7 were treated with or without AICAR for 12 hrs . R388 methylation of immunoprecipitated SIRT7 was determined by western blotE - F . AICAR reduces the percentage of R388 - methylated endogenous SIRT7 in MEF cells . R388 - methylated endogenous SIRT7 was captured from control and AICAR - treated MEF cells by using R388 site - specific methylation antibody ( E ) . Input , output , and immunoprecipitate were analyzed by western blot to quantify the percentage of R388 - methylated protein ( F ) ( mean ± SD , n = 3 experimental replicates ) G . AMPK deletion increases SIRT7 R388 methylation . Flag - tagged SIRT7 was stably expressed in Ampk wildtype ( WT ) and Ampk α1 / α2 double - knockout ( DKO ) MEF cells and 293A cells . SIRT7 protein was immunopurified with Flag - beads , R388 methylation was determined by western blotH . AMPK is required for glucose starvation - induced R388 hypomethylation of SIRT7 . Ampk wildtype ( WT ) and Ampk α1 / α2 double - knockout ( DKO ) MEF cells stably expressing Flag - SIRT7 were cultured with or without glucose for 12 hrs . SIRT7 was purified with Flag - beads and R388 methylation was determined by western blotI . PRMT6 is necessary for AMPK - induced SIRT7 hypomethylation . Flag - tagged SIRT7 was stably expressed in scramble control MEF cells or cells expressing two independent shRNAs targeting Prmt6 ( # 1 and # 2 ) . Cells were cultured with or without glucose for 12 hrs . R388 methylation of immunopurified SIRT7 was determined by western blotJ . PRMT6 is necessary for AMPK - induced SIRT7 hypomethylation . Wildtype MEFs and Ampk DKO cells were treated with PRMT6i for 24 hrs . SIRT7 was purified by Flag - beads , and R388 methylation of SIRT7 was determined by western blotK . Both AICAR treatment and glucose depletion impair the association between Prmt6 and Sirt7 . MEF cells were cultured with or without glucose and treated with AICAR as indicated . Co - immunoprecipitation was performed using SIRT7 antibody . Input and immunoprecipitates were analyzed by western blotL . R388 methylation of SIRT7 does not modulate its phosphorylation . MEF cells stably expressing Flag - tagged wildtype SIRT7 or RK mutant were treated with PRMT6i ( 24 hrs ) and cultured with or without glucose ( 12 hrs ) as indicated . SIRT7 protein was immunopurified with Flag - beads , R388 methylation and phosphorylation of SIRT7 were determined by western

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Figure 5A. Glucose depletion reduces SIRT7 methylation. MEF cells stably expressing Flag-SIRT7 were cultured in medium depleted with glucose (12 hrs), FBS (12 hrs) or glutamine (24 hrs). R388 methylation of immunopurified SIRT7 and Ampk phosphorylation were determined by western blot. R388 methylation level was normalized to Flag-SIRT7 (Ratio)B. Glucose starvation decreases R388 methylation of SIRT7 in a time-dependent manner. MEF cells stably expressing Flag-SIRT7 were cultured in glucose-free media as indicated. R388 methylation level of immunopurified SIRT7 was determined by western blotC. Glucose dose-dependently upregulates R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were cultured with increasing concentrations of glucose as indicated for 12 hrs. R388 methylation of SIRT7 and Ampk phosphorylation were determined by western blotD. AICAR treatment decreases R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were treated with or without AICAR for 12 hrs. R388 methylation of immunoprecipitated SIRT7 was determined by western blotE-F. AICAR reduces the percentage of R388-methylated endogenous SIRT7 in MEF cells. R388-methylated endogenous SIRT7 was captured from control and AICAR-treated MEF cells by using R388 site-specific methylation antibody (E). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (F) (mean±SD, n=3 experimental replicates)G. AMPK deletion increases SIRT7 R388 methylation. Flag-tagged SIRT7 was stably expressed in Ampk wildtype (WT) and Ampk α1/α2 double-knockout (DKO) MEF cells and 293A cells. SIRT7 protein was immunopurified with Flag-beads, R388 methylation was determined by western blotH. AMPK is required for glucose starvation-induced R388 hypomethylation of SIRT7. Ampk wildtype (WT) and Ampk α1/α2 double-knockout (DKO) MEF cells stably expressing Flag-SIRT7 were cultured with or without glucose for 12 hrs. SIRT7 was purified with Flag-beads and R388 methylation was determined by western blotI. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Flag-tagged SIRT7 was stably expressed in scramble control MEF cells or cells expressing two independent shRNAs targeting Prmt6 (#1 and #2). Cells were cultured with or without glucose for 12 hrs. R388 methylation of immunopurified SIRT7 was determined by western blotJ. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Wildtype MEFs and Ampk DKO cells were treated with PRMT6i for 24 hrs. SIRT7 was purified by Flag-beads, and R388 methylation of SIRT7 was determined by western blotK. Both AICAR treatment and glucose depletion impair the association between Prmt6 and Sirt7. MEF cells were cultured with or without glucose and treated with AICAR as indicated. Co-immunoprecipitation was performed using SIRT7 antibody. Input and immunoprecipitates were analyzed by western blotL. R388 methylation of SIRT7 does not modulate its phosphorylation. MEF cells stably expressing Flag-tagged wildtype SIRT7 or RK mutant were treated with PRMT6i (24 hrs) and cultured with or without glucose (12 hrs) as indicated. SIRT7 protein was immunopurified with Flag-beads, R388 methylation and phosphorylation of SIRT7 were determined by western blot


Figure 6Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte . After fasting for 16 hrs , mice were injected with glucose . Hepatic tissue was harvested in a time - dependent manner as indicated ( n = 3 for each time point ) . SIRT7 protein was immunoprecipitated from whole liver extracts . R388 methylation of Sirt7 was determined by western blot ( A Scale bar in the upper panel , 500 µm ; scale bar in the lower panel , 100 µmGlucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte . After fasting for 16 hrs , mice were injected with glucose . Hepatic tissue was harvested in a time - dependent manner as indicated ( n = 3 for each time point ) . SIRT7 protein was immunoprecipitated from whole liver extracts . R388 methylation of Sirt7 was determined b immunohistochemical staining ( B ) . Scale bar in the upper panel , 500 µm ; scale bar in the lower panel , 100 µmAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs . R388 methylation of SIRT7 was determined by western blot ( C ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs H3K18ac level at SIRT7 - target gene promoters were determined by ChIP - qPCR . Normal rabbit IgG was used as a negative control ( D ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs mRNA expression was determined by qPCR analysis ( E ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs Mitochondria mass was determined by Mito Tracker Red staining ( F ) . ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t

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Figure 6Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined by western blot (A Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µmGlucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined b immunohistochemical staining (B). Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µmAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs. R388 methylation of SIRT7 was determined by western blot (C) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs H3K18ac level at SIRT7-target gene promoters were determined by ChIP-qPCR. Normal rabbit IgG was used as a negative control (D) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs mRNA expression was determined by qPCR analysis (E) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs Mitochondria mass was determined by Mito Tracker Red staining (F). (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test


Figure 1B . Normalized fitness functions of E . coli BW25113 in the absence of tet ( B ) and their corresponding model fits in the presence of varying concentrations of antibiotics TET , DOX , and MCN in the media ( [ S ] M ) . Normalized quadruplicate data is shown for each antibiotic .

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Figure 1B. Normalized fitness functions of E. coli BW25113 in the absence of tet(B) and their corresponding model fits in the presence of varying concentrations of antibiotics TET, DOX, and MCN in the media ([S]M). Normalized quadruplicate data is shown for each antibiotic.


Figure 2B . Normalized fitness functions of cells expressing wild type tet ( B ) and their corresponding model fits in the presence of TET , DOX and MCN . Triplicate data is shown for each

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Figure 2B. Normalized fitness functions of cells expressing wild type tet(B) and their corresponding model fits in the presence of TET, DOX and MCN. Triplicate data is shown for each antibiotic


Figure 3B . Model fits for E . coli BW25113 strains expressing a chromosomal copy of tet ( B ) WT and seven variants in TET , DOX , and MCN . Our model fit triplicate fitness function data for all strains ( Figure S2 ) . At low drug concentrations for all antibiotics , tet ( B ) G366C exhibits much slower growth rates than wild - type . Compared to wild - type , strains expressing tet ( B ) L9F exhibit similar growth rates at low and high drug concentrations but have faster growth rates at intermediate drug concentrations . C . Fitness landscapes with mutants overlaid for TET = 35 µg / mL , DOX = 10 µg / mL , and MCN = 2 . 5 µg / mL corresponding to the dashed lines in 3B .

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Figure 3B. Model fits for E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT and seven variants in TET, DOX, and MCN. Our model fit triplicate fitness function data for all strains (Figure S2). At low drug concentrations for all antibiotics, tet(B)G366C exhibits much slower growth rates than wild-type. Compared to wild-type, strains expressing tet(B)L9F exhibit similar growth rates at low and high drug concentrations but have faster growth rates at intermediate drug concentrations.C. Fitness landscapes with mutants overlaid for TET=35 µg/mL, DOX=10 µg/mL, and MCN=2.5 µg/mL corresponding to the dashed lines in 3B.


Figure 4E . coli BW25113 strains expressing a chromosomal copy of tet ( B ) WT or variant were grown in 2 % arabinose supplemented LB in the absence of drug and harvested at mid - exponential phase . Membrane samples were prepared from three individual colonies grown independently ( Figures S5 and S6 ) . Compared to wild - type , strains expressing tet ( B ) L9F consistently have higher in vivo protein levels where as strains expressing tet ( B ) V339C and tet ( B ) G366C have lower in vivo protein levels as detected by Western Blot analysis . All four variants located in the interdomain loop exhibited in vivo protein levels similar to wild - type .

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Figure 4E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT or variant were grown in 2% arabinose supplemented LB in the absence of drug and harvested at mid-exponential phase. Membrane samples were prepared from three individual colonies grown independently (Figures S5 and S6). Compared to wild-type, strains expressing tet(B)L9F consistently have higher in vivo protein levels where as strains expressing tet(B)V339C and tet(B)G366C have lower in vivo protein levels as detected by Western Blot analysis. All four variants located in the interdomain loop exhibited in vivo protein levels similar to wild-type.


Figure 5A . The spread of κ and γ fits for 1000 randomly selected parameters A and B from a set gamma distribution shows a linear correlation between κ and γ . B . The calculated mean and error of γ / κ for the sampled parameter fittings shown in Figure S2 . The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $ \ frac { \ gamma } { \ kappa } \ \ approx k _ { 1 } \ frac { { 2P } _ { \ text { tot } } } { D _ { 2 } } $ .

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Figure 5A. The spread of κ and γ fits for 1000 randomly selected parameters A and B from a set gamma distribution shows a linear correlation between κ and γ.B. The calculated mean and error of γ/κ for the sampled parameter fittings shown in Figure S2. The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $\frac{\gamma}{\kappa}\ \approx k_{1}\frac{{2P}_{\text{tot}}}{D_{2}}$.


Figure 6A . Changes in drug binding affinity Δk1 and pumping efficiency Δr for TetB variants relative to wild type in TET , DOX and MCN . Error bars are comprised from a propagation of errors from A and B global parameter modeling and in vivo protein level determination .

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Figure 6A. Changes in drug binding affinity Δk1 and pumping efficiency Δr for TetB variants relative to wild type in TET, DOX and MCN. Error bars are comprised from a propagation of errors from A and B global parameter modeling and in vivo protein level determination.


Figure 7A . Triplicate normalized fitness functions ( * ) for E . coli BW25113 strains harboring plasmid copies of tet ( B ) WT , tet ( B ) K183E , A290V , T326I ( variant 6 ) and tet ( B ) F151I , F179L , T326A ( variant 7 ) in TET and their corresponding least squares model fits ( solid line ) where both variants display decreased fitness in TET compared to WT .

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Figure 7A. Triplicate normalized fitness functions (*) for E. coli BW25113 strains harboring plasmid copies of tet(B)WT, tet(B)K183E,A290V,T326I (variant 6) and tet(B)F151I,F179L,T326A (variant 7) in TET and their corresponding least squares model fits (solid line) where both variants display decreased fitness in TET compared to WT.


Figure 1 ( B ) Representative images of mCherry - optoYAP in HEK293T cells imaged with only 561 nm laser following the protocol in ( A ) . Scale bars , 10 μm . ( B ' ) Fold - change in nuclear localisation of mCherry - optoYAP ( n = 50 cells from 3 independent experiments ) . ( C ) Representative images of the same cells in ( B ) exposed to both 488 and 561 nm following the protocol shown in ( A ) . Scale bars , 10 μm . ( C ' ) Fold - change in nuclear localisation of the mCherry - optoYAP ( n = 50 cells from 3 independent experiments ) . ( D ) HEK293T cells transfected with optoYAP were subjected to three cycles of activation protocol and recovery in the dark . ( Top ) The ratio of mCherry - optoYAP signal in the nucleus to the cytoplasmic mCherry - optoYAP signal ( n = 8 cells from 2 independent experiments ) . ( Bottom ) Representative images of the mCherry - optoYAP signal from the same HEK293T cells at each cycle . Scale bars , 10 μm .

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Figure 1(B) Representative images of mCherry-optoYAP in HEK293T cells imaged with only 561 nm laser following the protocol in (A). Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optoYAP (n=50 cells from 3 independent experiments).(C) Representative images of the same cells in (B) exposed to both 488 and 561 nm following the protocol shown in (A). Scale bars, 10 μm. (C') Fold-change in nuclear localisation of the mCherry-optoYAP (n=50 cells from 3 independent experiments).(D) HEK293T cells transfected with optoYAP were subjected to three cycles of activation protocol and recovery in the dark. (Top) The ratio of mCherry-optoYAP signal in the nucleus to the cytoplasmic mCherry-optoYAP signal (n=8 cells from 2 independent experiments). (Bottom) Representative images of the mCherry-optoYAP signal from the same HEK293T cells at each cycle. Scale bars, 10 μm.


Figure 3 ( B ) Example images of embryos expressing optofYap ( mCherry - tagged ) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf . Scale bars , 10 μm . ( B ' ) Fold - change in nuclear localisation of mCherry - optofYap ( n = 20 cells from 3 independent experiments ) .

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Figure 3(B) Example images of embryos expressing optofYap (mCherry-tagged) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf. Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optofYap (n=20 cells from 3 independent experiments).


Figure 4Soft agar colony formation assay for HFF ( A - B ) cells respectively grown on soft agar for one week under different light conditions as described in Fig . 1A . Scale bars , 1 mm . Representative images of colony growth in HFF cells respectively under different activation conditions . Diameter of individual colonies formed under different light conditions and genetic backgrounds . Colonies were counted and measured from 3 biological replicates . Soft agar colony formation assay for MKN28 ( C - D ) cells respectively grown on soft agar for one week under different light conditions as described in Fig . 1A . Scale bars , 1 mm . Representative images of colony growth in MKN28 cells respectively under different activation conditions . Diameter of individual colonies formed under different light conditions and genetic backgrounds . Colonies were counted and measured from 3 biological replicates .

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Figure 4Soft agar colony formation assay for HFF (A-B) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in HFF cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.Soft agar colony formation assay for MKN28 (C-D) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in MKN28 cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.


Figure 1Time - dependent acceptor fluorescence changes due to FRET during co - translational insertion of acceptor - labeled LepB75 into donor - labeled translocon .

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Figure 1Time-dependent acceptor fluorescence changes due to FRET during co-translational insertion of acceptor-labeled LepB75 into donor-labeled translocon.


Figure 2Analysis of LepB94 translation products on SDS - PAGE . Translation products are visualized by the fluorescence of N - terminal Atto655 . Pausing intermediates are indicated P1 and P2 . Fitting of time courses ( filled circles ) of the translation of LepB mRNA constructs of increasing length ( Appendix Fig S3 ) by delay - exponential functions ( red lines ) . Stopped - flow time courses of co - translational LepB insertion into SecYEG ( Atto488 donor - in at position 111 of SecY ) , as monitored by the fluorescence of the Atto655 acceptor placed at the N - terminus of the nascent peptide . Fits ( red lines ) were obtained by global fitting Intrinsic fluorescence intensities ( IFI ) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting ( red squares ) . Confidence contours ( shaded areas ) were calculated using the Fitspace

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Figure 2Analysis of LepB94 translation products on SDS-PAGE. Translation products are visualized by the fluorescence of N-terminal Atto655. Pausing intermediates are indicated P1 and P2. Fitting of time courses (filled circles) of the translation of LepB mRNA constructs of increasing length (Appendix Fig S3) by delay-exponential functions (red lines).  Stopped-flow time courses of co-translational LepB insertion into SecYEG (Atto488 donor-in at position 111 of SecY), as monitored by the fluorescence of the Atto655 acceptor placed at the N-terminus of the nascent peptide. Fits (red lines) were obtained by global fitting Intrinsic fluorescence intensities (IFI) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting (red squares). Confidence contours (shaded areas) were calculated using the Fitspace algorithm


Figure 3 Time course of EmrD135 translation . Translation was monitored by SDS - PAGE as in Fig 2A . Time course of EmrD ( - ) 135 translation . Stopped - flow time courses for translocon insertion of TM1 of EmrD ( black ) and EmrD ( - ) ( blue ) with mRNA constructs of varying chain length ( 50 to 135 aa ) . The FRET acceptor ( Atto655 ) was placed at the N - terminus of the nascent peptide , the donor ( Atto488 ) at position 111 ( donor - in ) of SecY . The results of global fitting are indicated ( red lines ) . Intrinsic fluorescence intensities ( IFIs ) calculated for EmrD ( black ) and EmrD ( - ) ( blue ) . Error margins ( shaded areas ) are calculated using the Fitspace algorithmStopped - flow traces for co - translational insertion of LepB94 ( red , from Fig 2 ) , EmrD135 ( black , from Fig 4E ) , and EmrD ( - ) 135 ( blue , from Fig 4E ) , rescaled according to the translation efficiency .

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Figure 3 Time course of EmrD135 translation. Translation was monitored by SDS-PAGE as in Fig 2A. Time course of EmrD(-)135 translation.  Stopped-flow time courses for translocon insertion of TM1 of EmrD (black) and EmrD(-) (blue) with mRNA constructs of varying chain length (50 to 135 aa).The FRET acceptor (Atto655) was placed at the N-terminus of the nascent peptide, the donor (Atto488) at position 111 (donor-in) of SecY. The results of global fitting are indicated (red lines). Intrinsic fluorescence intensities (IFIs) calculated for EmrD (black) and EmrD(-) (blue). Error margins (shaded areas) are calculated using the Fitspace algorithmStopped-flow traces for co-translational insertion of LepB94 (red, from Fig 2), EmrD135 (black, from Fig 4E), and EmrD(-)135 (blue, from Fig 4E), rescaled according to the translation efficiency.


Figure 4 A , B , C LepB ( A ) , EmrD ( B ) , and EmrD ( - ) ( C ) are translated for the times indicated and subjected to PK treatment ( Materials and Methods ) . Sensitivity to PK in the absence of SecYEG ( black circles ) or in the presence of SecYEG ( red squares ) , and co - translational translocon insertion monitored by FRET ( blue trace ) are depicted . Error bars represent standard deviations ( n ≥ 3 ) and the corresponding lines represent fits to delay - exponential functions .

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Figure 4 A, B, C LepB (A), EmrD (B), and EmrD(-) (C) are translated for the times indicated and subjected to PK treatment (Materials and Methods). Sensitivity to PK in the absence of SecYEG (black circles) or in the presence of SecYEG (red squares), and co-translational translocon insertion monitored by FRET (blue trace) are depicted. Error bars represent standard deviations (n≥3) and the corresponding lines represent fits to delay-exponential functions.


Figure 2DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB IL - 2nil ( 32694 ) ordered by increasing fold change of tag count for TB IL - 2 compared to TB IL - 2nil . The sequence tag density is also shown in parallel at the same sites for the DNase - Seq analyses of CD4 naïve T cells ( TN ) , TB IL - 2 DMSO and TB IL - 2 Ruxolitinib and ATAC samples from in vivo derived Th2 ( Shih et al . , 2016 ) , Th1 and Th17 cells . The location of the 1000 IL - 2 pDHSs is shown to the right . Average DNase - Seq tag density profile for the 1000 IL - 2 pDHSs in both replicates of TB IL - 2 and TB IL - 2nil samples , and for naïve T cells ( TN ) . Number of IL - 2 pDHSs which are detected in in vivo purified CD4 T cell subsets ( Chen et al . , 2020 , Garg et al . , 2019 , Shih et al . , 2016 ) . UCSC genome browser tracks with examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the Rad50 gene which controls the Th2 cytokines Il13 , Il4 and Il5 , plus IL - 2 pDHSs within the Tnfrsf9 and Cish loci . IL - 2 pDHSs are highlighted with blue boxes .

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Figure 2DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL-2nil (32694) ordered by increasing fold change of tag count for TB IL-2 compared to TB IL-2nil. The sequence tag density is also shown in parallel at the same sites for the DNase-Seq analyses of CD4 naïve T cells (TN), TB IL-2 DMSO and TB IL-2 Ruxolitinib and ATAC samples from in vivo derived Th2 (Shih et al., 2016), Th1 and Th17 cells. The location of the 1000 IL-2 pDHSs is shown to the right.Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-2 and TB IL-2nil samples, and for naïve T cells (TN).Number of IL-2 pDHSs which are detected in in vivo purified CD4 T cell subsets (Chen et al., 2020, Garg et al., 2019, Shih et al., 2016).UCSC genome browser tracks with examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci. IL-2 pDHSs are highlighted with blue boxes.


Figure 3Jund mRNA levels in TB IL - 2 and TB IL - 2nil . Standard deviation is shown from the mean of 3 samples . P - values were calculated from the RNA - seq data using Limma with Benjamini - Hochberg correction for multiple testing . Western blot analyses showing JUND and pSTAT5 protein levels in TB IL - 2 and TB IL - 2nil ( left panel ) with the signal calculated from 2 replicates relative to TB IL - 2 in the right panel . DNA motif density plots ( right ) showing the location of binding motifs for RUNX , ETS , STAT and AP - 1 transcription factors at the 32694 DHSs detected in TB IL - 2 and TB IL - 2nil , as depicted in Figure 2A , ordered according to fold enrichment of DNase - Seq tag counts in TB IL - 2 compared to TB IL - 2nil ( left ) . DNA sequence tag density plots from ChIP - Seq assays of RUNX1 , ETS1 , STAT5 and JUND in TB IL - 2 , and STAT5 and JUND in TB IL - 2nil ordered as in ( D ) . UCSC genome browser tracks at the Il13 / Il4 / Il5 Th2 cytokine locus LCR ( indicated by the grey box within the Rad50 gene ) , Gzmb and Il24 showing DNase - Seq and ChIP - Seq for STAT5 and JUND in TB IL - 2 and TB IL - 2nil . Replicate JUND and STAT5 ChIP - Seq tracks are shown for TB IL - 2 .

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Figure 3Jund mRNA levels in TB IL-2 and TB IL-2nil. Standard deviation is shown from the mean of 3 samples. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.Western blot analyses showing JUND and pSTAT5 protein levels in TB IL-2 and TB IL-2nil (left panel) with the signal calculated from 2 replicates relative to TB IL-2 in the right panel.DNA motif density plots (right) showing the location of binding motifs for RUNX, ETS, STAT and AP-1 transcription factors at the 32694 DHSs detected in TB IL-2 and TB IL-2nil, as depicted in Figure 2A, ordered according to fold enrichment of DNase-Seq tag counts in TB IL-2 compared to TB IL-2nil (left).DNA sequence tag density plots from ChIP-Seq assays of RUNX1, ETS1, STAT5 and JUND in TB IL-2, and STAT5 and JUND in TB IL-2nil ordered as in (D).UCSC genome browser tracks at the Il13/Il4/Il5 Th2 cytokine locus LCR (indicated by the grey box within the Rad50 gene), Gzmb and Il24 showing DNase-Seq and ChIP-Seq for STAT5 and JUND in TB IL-2 and TB IL-2nil. Replicate JUND and STAT5 ChIP-Seq tracks are shown for TB IL-2.


Figure 4DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB + IL - 2 ( A , 38645 ) and in TB + IL - 2 and TB + IL - 2nil ( B , 36067 ) , ordered by increasing fold change in tag count as indicated . The position of the 1015 IL - 2 - dependent inducible DHSs is shown alongside . Average DHS signal at the 1015 IL - 2 - dependent inducible DHSs in TB IL - 2 , TB + IL - 2 , TB + IL - 2nil and naïve T cells stimulated with PMA / I ( TN + ) . Bar graph showing the number of IL - 2 iDHSs found within 25 kb of an IL2 pDHS . Examples of IL - 2 iDHSs which are proximal to IL - 2 pDHSs at the Csf2 / Il2 and Il10 loci . IL - 2 iDHSs are highlighted in red and IL - 2 pDHSs in blue .

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Figure 4DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB+ IL-2 (A, 38645) and in TB+ IL-2 and TB+ IL-2nil (B, 36067), ordered by increasing fold change in tag count as indicated. The position of the 1015 IL-2-dependent inducible DHSs is shown alongside.Average DHS signal at the 1015 IL-2-dependent inducible DHSs in TB IL-2, TB+ IL-2, TB+ IL-2nil and naïve T cells stimulated with PMA/I (TN+).Bar graph showing the number of IL-2 iDHSs found within 25 kb of an IL2 pDHS.Examples of IL-2 iDHSs which are proximal to IL-2 pDHSs at the Csf2/Il2 and Il10 loci. IL-2 iDHSs are highlighted in red and IL-2 pDHSs in blue.


Figure 5Hierarchical clustering of RNA - Seq data from 3 replicates as described in ( A ) . MA plot showing the genes differentially regulated between TB IL - 2nil and TB IL - 2 . The number of genes with a fold change > 3 and adj p - value < 0 . 05 are highlighted . P - values were calculated from the RNA - seq data using Limma with Benjamini - Hochberg correction for multiple testing . log2 fold change in gene expression in TB IL - 2nil compared to TB IL - 2 for the nearest gene to the DHSs ranked according to the fold change in DNase - Seq tag count for TB IL - 2 / TB IL - 2nil . The DNase - Seq tag density plots are taken from Figure 2A for TB IL - 2 , TB IL - 2nil and TN . UCSC genome browser tracks showing DNase - seq and RNA - seq at the Socs1 ( E ) and Sell loci ( F ) .

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Figure 5Hierarchical clustering of RNA-Seq data from 3 replicates as described in (A).MA plot showing the genes differentially regulated between TB IL-2nil and TB IL-2. The number of genes with a fold change >3 and adj p-value <0.05 are highlighted. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.log2 fold change in gene expression in TB IL-2nil compared to TB IL-2 for the nearest gene to the DHSs ranked according to the fold change in DNase-Seq tag count for TB IL-2/TB IL-2nil. The DNase-Seq tag density plots are taken from Figure 2A for TB IL-2, TB IL-2nil and TN.UCSC genome browser tracks showing DNase-seq and RNA-seq at the Socs1 (E) and Sell loci (F).


Figure 6Relative mRNA expression levels in TB + IL - 2 and TB + IL - 2nil ( A ) and TB + DMSO and TB + Ruxolitinib ( B ) . The expression level in the IL - 2 / DMSO TB + sample is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - test with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . ) UCSC genome browser tracks showing DNase - Seq and RNA - Seq at the IL - 2 regulated genes Csf2 / Il3 and at the LCR within the Rad50 gene ( grey box ) which controls the Th2 cytokines Il13 , Il4 and Il5 . IL - 2 pDHSs are highlighted in blue and IL - 2 iDHSs in red .

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Figure 6Relative mRNA expression levels in TB+ IL-2 and TB+ IL-2nil (A) and TB+ DMSO and TB+ Ruxolitinib (B). The expression level in the IL-2/DMSO TB+ sample is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)UCSC genome browser tracks showing DNase-Seq and RNA-Seq at the IL-2 regulated genes Csf2/Il3 and at the LCR within the Rad50 gene (grey box) which controls the Th2 cytokines Il13, Il4 and Il5. IL-2 pDHSs are highlighted in blue and IL-2 iDHSs in red.


Figure 7DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB IL ‑ 2nil ( data taken from Figure 2A ) , duplicate DNase - seq samples for TB IL - 7 and TB IL - 7nil are shown in parallel for the same sites . Average DNase - Seq tag density profile for the 1000 IL - 2 pDHSs in both replicates of TB IL - 7 and TB IL - 7nil . UCSC genome browser tracks with DNase - Seq data for TB IL - 7 and TB IL - 7nil showing examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the Rad50 gene which controls the Th2 cytokines Il13 , Il4 and Il5 , plus IL - 2 pDHSs within the Tnfrsf9 and Cish loci ( C ) IL - 2 pDHSs are highlighted with blue boxes . UCSC genome browser tracks with DNase - Seq data for TB IL - 7 and TB IL - 7nil showing examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the non - primed control regions from the CD3 gene cluster and the Tbp locus ( D ) . IL - 2 pDHSs are highlighted with blue boxes . Average DHS signal at the 1015 IL - 2 regulated inducible DHSs in TB + IL - 7 and TB + IL - 7nil . Relative mRNA expression levels in TB + IL - 7 and TB + IL - 7nil . The expression level in the TB + IL - 7 is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - test with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . )

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Figure 7DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL‑2nil (data taken from Figure 2A), duplicate DNase-seq samples for TB IL-7 and TB IL-7nil are shown in parallel for the same sites.Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-7 and TB IL-7nil.UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci (C) IL-2 pDHSs are highlighted with blue boxes.UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the non-primed control regions from the CD3 gene cluster and the Tbp locus (D). IL-2 pDHSs are highlighted with blue boxes.Average DHS signal at the 1015 IL-2 regulated inducible DHSs in TB+ IL-7 and TB+ IL-7nil.Relative mRNA expression levels in TB+ IL-7 and TB+ IL-7nil. The expression level in the TB+ IL-7 is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)


Figure 8Flow cytometric analysis of IL - 7Rα expression by 2W1S - specific CD44hi CD4 T cells in the presence or absence of cre recombinase . Average number of 2W1S - specific cells recovered / mouse after sorting 3 replicate samples of Il7rf / f compared to CD4creERT2 x Il7rf / f . Errors bars indicate the standard deviation . Average ATAC - Seq signal at the 411 IL - 2 pDHSs which contain a STAT binding motif . UCSC genome browser tracks at loci with IL - 2 pDHSs showing replicate ATAC - Seq samples for CD4creERT2 x Il7rf / f memory T cells , Il7rf / f control memory T cells , replicate DNase - Seq samples for TB IL - 2 and TB IL - 2nil and TB IL - 2 and TB IL ‑ 2nil STAT5 ChIP - Seq samples . The grey boxes signify IL - 2 pDHSs which diminish after disruption of the Il7r gene .

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Figure 8Flow cytometric analysis of IL-7Rα expression by 2W1S-specific CD44hi CD4 T cells in the presence or absence of cre recombinase.Average number of 2W1S-specific cells recovered/mouse after sorting 3 replicate samples of Il7rf/f compared to CD4creERT2 x Il7rf/f . Errors bars indicate the standard deviation.Average ATAC-Seq signal at the 411 IL-2 pDHSs which contain a STAT binding motif.UCSC genome browser tracks at loci with IL-2 pDHSs showing replicate ATAC-Seq samples for CD4creERT2 x Il7rf/f memory T cells, Il7rf/f control memory T cells, replicate DNase-Seq samples for TB IL-2 and TB IL-2nil and TB IL-2 and TB IL‑2nil STAT5 ChIP-Seq samples. The grey boxes signify IL-2 pDHSs which diminish after disruption of the Il7r gene.


Figure 9UCSC genome browser tracks showing ChIP - Seq and DNase - Seq at the LCR within the Rad50 gene which controls the Il4 / Il13 / Il5 Th2 cytokine locus , and also Il17a , Il12rb2 and Tbx21 . The grey boxes indicate IL - 2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages .

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Figure 9UCSC genome browser tracks showing ChIP-Seq and DNase-Seq at the LCR within the Rad50 gene which controls the Il4/Il13/Il5 Th2 cytokine locus, and also Il17a, Il12rb2 and Tbx21. The grey boxes indicate IL-2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages.


Figure 10mRNA expression levels of JUN family transcription factors . Normalised TPM is shown for TB IL - 2 , TB IL2nil , TB + IL - 2 and TB + IL - 2nil . Standard deviation is shown for 3 replicates . Average JUNB ChIP - Seq signal at the 1000 IL - 2 pDHSs in TB IL - 2 and TB + IL - 2 ( Bevington et al . , 2016 ) . Average JUND ChIP - Seq signal at the 1000 IL - 2 pDHSs in Junb + / + CD4Cre ( Junb WT ) and Junbf / fCD4Cre ( Junb KO ) Th17 cells . Genome browser tracks showing open chromatin ( DNase - Seq or ATAC - Seq ) and ChIP - Seq in Th0 , Th1 and Th17 cells at the Il17a locus ( Carr et al . , 2017 , Ciofani et al . , 2012 , Vahedi et al . , 2012 ) . The grey box indicates a DHS which binds JUND . Fold change in gene expression in TB + IL - 2 cells for Jund - / - compared to Jund + / + mice . Data from 2 replicates are shown . Genome browser tracks showing open chromatin ( DNase - Seq or ATAC - Seq ) and ChIP - Seq in Th0 , Th1 and Th17 cells at the Irf8 locus . The grey box indicates an IL - 2 pDHS which binds JUND .

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Figure 10mRNA expression levels of JUN family transcription factors. Normalised TPM is shown for TB IL-2, TB IL2nil, TB+ IL-2 and TB+ IL-2nil. Standard deviation is shown for 3 replicates.Average JUNB ChIP-Seq signal at the 1000 IL-2 pDHSs in TB IL-2 and TB+ IL-2 (Bevington et al., 2016).Average JUND ChIP-Seq signal at the 1000 IL-2 pDHSs in Junb+/+CD4Cre (Junb WT) and Junbf/fCD4Cre (Junb KO) Th17 cells.Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Il17a locus (Carr et al., 2017, Ciofani et al., 2012, Vahedi et al., 2012). The grey box indicates a DHS which binds JUND.Fold change in gene expression in TB+ IL-2 cells for Jund -/- compared to Jund+/+ mice. Data from 2 replicates are shown.Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Irf8 locus. The grey box indicates an IL-2 pDHS which binds JUND.


Figure 1A , B . A missense mutation in SPOP gene ( Q165P , T > G ) was identified in a PCa patient . Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation ( A ) whereas the liver metastasis harbored a homozygous Q165P mutation ( B ) . D , E . IHC analysis of BRD4 protein expression in SPOP wild type ( WT ) and mutated ( MUT ) PCa patient samples . The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in ( D ) and the quantified IHC data are shown in ( E ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The red dot in ( E ) indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) . F , G . IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples . The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in ( F ) and the quantified IHC data are shown in ( G ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The green dot indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) .

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Figure 1A, B. A missense mutation in SPOP gene (Q165P, T >G) was identified in a PCa patient. Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation (A) whereas the liver metastasis harbored a homozygous Q165P mutation (B).D, E. IHC analysis of BRD4 protein expression in SPOP wild type (WT) and mutated (MUT) PCa patient samples. The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in (D) and the quantified IHC data are shown in (E). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The red dot in (E) indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).F, G. IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples. The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in (F) and the quantified IHC data are shown in (G). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The green dot indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; *** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).


Figure 2LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP ( A ) LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination ( B ) . C . C4 - 2 cells were stably infected with the lenti - virus expressing empty vector ( EV ) or SPOP mutants and harvested for western blot analysis with the indicated antibodies . D . C4 - 2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies . T1 : tumor 1 ; T2 : tumor 2 . E . Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields .

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Figure 2LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP (A)LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination (B).C. C4-2 cells were stably infected with the lenti-virus expressing empty vector (EV) or SPOP mutants and harvested for western blot analysis with the indicated antibodies.D. C4-2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies. T1: tumor 1; T2: tumor 2.E. Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors. Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields.


Figure 3Co - IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant ( F ) Co - IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P ( G ) .

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Figure 3Co-IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant (F)Co-IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P (G).


Figure 4A . DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis . B . MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 . Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells . The survival curve showed IC50 for SPOP Q165P ( 0 . 69 µM ) and EV cells ( 1 . 08 µM ) ( C ) . DU145 cells expressing lenti - EV or lenti - HA - SPOP - Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantitative data shown in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 . F . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody . The enrichment of BRD4 at c - MYC enhancer and RAC1 promoter was analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 . G . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac - H3 antibody . The level of Ac - H3 at c - MYC enhancer and RAC1 promoter is analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 .

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Figure 4A. DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis.B. MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells. The survival curve showed IC50 for SPOP Q165P (0.69 µM) and EV cells (1.08 µM) (C).DU145 cells expressing lenti-EV or lenti-HA-SPOP-Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantitative data shown in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01.F. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody. The enrichment of BRD4 at c-MYC enhancer and RAC1 promoter was analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01.G. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac-H3 antibody. The level of Ac-H3 at c-MYC enhancer and RAC1 promoter is analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01.


Figure 5B , C . SCID male mice with PDX tumors were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) , CPI - 637 ( 30 mg / kg ) , combination of JQ1 ( 50 mg / kg ) and CPI - 637 ( 30 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of drug treatment were photographed ( B ) and tumor growth are shown in ( C ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 001 . PDX tumors in each treatment group were harvested for protein For western blot analysis , three tumors each group ( n = 3 ) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in ( D ) . PDX tumors in each treatment group were harvested for RNA extraction . For RT - qPCR , three tumors each group ( n = 3 ) were extracted RNA for RT - qPCR in ( E ) . Data are shown as means ± SEM . The P value comparing vehicle with each treatment group was calculated by unpaired two - tailed Student ' s t - test and indicated in the graph .

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Figure 5B, C. SCID male mice with PDX tumors were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), combination of JQ1 (50 mg/kg) and CPI-637 (30 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of drug treatment were photographed (B) and tumor growth are shown in (C). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; *** P < 0.001.PDX tumors in each treatment group were harvested for protein For western blot analysis, three tumors each group (n = 3) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in (D).PDX tumors in each treatment group were harvested for RNA extraction. For RT-qPCR, three tumors each group (n = 3) were extracted RNA for RT-qPCR in (E). Data are shown as means ± SEM. The P value comparing vehicle with each treatment group was calculated by unpaired two-tailed Student's t-test and indicated in the graph.


Figure 6Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for western blot analysis with the indicated antibodies ( A ) Asterisk , SRC3 at expected molecular mass . Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for IFC with p - AKT - S473 antibody ( B ) . E - cadherin antibody was used to indicate the cell membrane ( red ) and DAPI for nucleus ( blue ) . Scale bars : 25 µm . Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . The representative images of organoids after the treatment are shown in ( C ) Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . the quantified data of the organoid diameter are shown in ( D ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . E , F . 313HR ( SPOP WT ) and 573R ( Q165P ) organoids were cultured for 5 days , followed by the treatment with the indicated inhibitors for 5 days . The organoids were stained with Caspase - 3 antibody ( green ) to detect apoptotic cells . E - cadherin antibody staining was used to define the cell membrane ( red ) and the nucleus was counterstained with DAPI ( blue ) . The representative images of cleaved Caspase - 3 IFC staining are shown in ( E ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

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Figure 6Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for western blot analysis with the indicated antibodies (A) Asterisk, SRC3 at expected molecular mass.Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for IFC with p-AKT-S473 antibody (B). E-cadherin antibody was used to indicate the cell membrane (red) and DAPI for nucleus (blue). Scale bars: 25 µm.Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. The representative images of organoids after the treatment are shown in (C)Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. the quantified data of the organoid diameter are shown in (D). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.E, F. 313HR (SPOP WT) and 573R (Q165P) organoids were cultured for 5 days, followed by the treatment with the indicated inhibitors for 5 days. The organoids were stained with Caspase-3 antibody (green) to detect apoptotic cells. E-cadherin antibody staining was used to define the cell membrane (red) and the nucleus was counterstained with DAPI (blue). The representative images of cleaved Caspase-3 IFC staining are shown in (E). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


Figure 7A . C4 - 2 PCa cells infected with virus expressing EV , SPOP Q165P or F133V were harvested for western blot analysis . B MTS was performed to compare the cell growth between EV , Q165P , F133V C4 - 2 groups after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . C . Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4 - 2 cells . The survival curve showed IC50 for EV ( 1 . 01 µM ) , SPOP Q165P ( 0 . 621 µM ) and SPOP F133V cells ( 0 . 395 µM ) . D , E . C4 - 2 cells expressing EV , Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantification data in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 . F - H . A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration ( F ) . When the tumor reached 100 mm3 , mice were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of treatment were photographed ( G ) and tumor growth are shown in ( H ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 ; * * * P < 0 . 001 .

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Figure 7A. C4-2 PCa cells infected with virus expressing EV, SPOP Q165P or F133V were harvested for western blot analysis.B MTS was performed to compare the cell growth between EV, Q165P, F133V C4-2 groups after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.C. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4-2 cells. The survival curve showed IC50 for EV (1.01 µM), SPOP Q165P (0.621 µM) and SPOP F133V cells (0.395 µM).D, E. C4-2 cells expressing EV, Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantification data in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.F - H. A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration (F). When the tumor reached 100 mm3, mice were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of treatment were photographed (G) and tumor growth are shown in (H). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01; *** P < 0.001.


Figure 1 ( B ) Co - immunoprecipitation of HA - tagged Btz - WT or Btz - HD with Myc - tagged GFP or eIF4AIII from transfected S2R + cells using Myc - Trap Magnetic Agarose beads . The upper Western blot is blotted with anti - Myc and the lower with anti - HA . Input samples are shown on the left and immunoprecipitates on the right . Btz - WT co - immunoprecipitates with eIF4AIII , but Btz - HD does not , although the protein is equally stable . The asterisk represents a likely cleavage product of Btz . ( C - F ) show stage 10 egg chambers stained with anti - Vasa ( C - F ) or anti - GFP ( E ' , F ' ) . ( C ) wild type ; ( D ) germline clones homozygous for the null allele btz2 ; ( E ) btz2 germline clones rescued with Btz - WT ; ( F ) btz2 germline clones rescued with Btz - HD . Posterior is to the right . Scale bars , 80 μm . Vasa localization to the posterior pole of the oocyte is lost in btz mutants and is rescued by the wild type but not the EJC interaction - defective transgene . Wild - type Btz - GFP , but not the mutant form , is also slightly enriched at the posterior pole .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Co-immunoprecipitation of HA-tagged Btz-WT or Btz-HD with Myc-tagged GFP or eIF4AIII from transfected S2R+ cells using Myc-Trap Magnetic Agarose beads. The upper Western blot is blotted with anti-Myc and the lower with anti-HA. Input samples are shown on the left and immunoprecipitates on the right. Btz-WT co-immunoprecipitates with eIF4AIII, but Btz-HD does not, although the protein is equally stable. The asterisk represents a likely cleavage product of Btz.(C-F) show stage 10 egg chambers stained with anti-Vasa (C-F) or anti-GFP (E', F'). (C) wild type; (D) germline clones homozygous for the null allele btz2; (E) btz2 germline clones rescued with Btz-WT; (F) btz2 germline clones rescued with Btz-HD. Posterior is to the right. Scale bars, 80 μm. Vasa localization to the posterior pole of the oocyte is lost in btz mutants and is rescued by the wild type but not the EJC interaction-defective transgene. Wild-type Btz-GFP, but not the mutant form, is also slightly enriched at the posterior pole.


Figure 2 ( L - N ) electron micrographs showing cross - sections of larval muscle 6 in segment A3 , with the dorsal surface to the right . Scale bars , 10 μm . ( L ) btz2 / + ; ( M ) Df ( 3R ) BSC497 / + ; ( N ) btz2 / Df ( 3R ) BSC497 . Yellow arrows indicate examples of mitochondria interspersed between muscle fibers , and red arrows indicate mitochondria at the dorsal surface . Scale bars , 2 μm . ( O ) quantification of the ratio of the dorsal surface peak of ATP5 intensity to the second highest peak for the genotypes indicated . * * * , p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant by Mann - Whitney test . n = 9 ( btz / + ) , n = 11 ( Df / + , btz / Df ) , n = 6 ( btz / Df ; G14 > btz , C57 - GAL4 / + ) , n = 4 ( btz / Df ; Btz - WT , C57 > eIF4AIII RNAi ) , n = 5 ( btz / Df ; Btz - HD ) , n = 10 ( C57 > btz RNAi ) , or n = 3 ( C57 > mago RNAi ) . Error bars show mean ± SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(L-N) electron micrographs showing cross-sections of larval muscle 6 in segment A3, with the dorsal surface to the right. Scale bars, 10 μm. (L) btz2/+; (M) Df(3R)BSC497/+; (N) btz2/ Df(3R)BSC497. Yellow arrows indicate examples of mitochondria interspersed between muscle fibers, and red arrows indicate mitochondria at the dorsal surface. Scale bars, 2 μm.(O) quantification of the ratio of the dorsal surface peak of ATP5 intensity to the second highest peak for the genotypes indicated. ***, p<0.001; **p<0.01; *p<0.05; ns, not significant by Mann-Whitney test. n=9 (btz/+), n=11 (Df/+, btz/Df), n=6 (btz/Df; G14>btz, C57-GAL4/+), n=4 (btz/Df; Btz-WT, C57>eIF4AIII RNAi), n=5 (btz/Df; Btz-HD), n=10 (C57>btz RNAi), or n=3 (C57>mago RNAi). Error bars show mean ± SEM.