Figure 4A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506 . Data information : Scale bar is 2 µm . C , D . Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A , B . Data information : In all plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison test for C and D significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E . Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25 . F . Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT . Data information : In all plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB in F Statistical significance was assessed using Student ' s t - test in F , significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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

Figure 4A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506. Data information: Scale bar is 2 µm.C, D. Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A, B. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test for C and D significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.E. Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25. F. Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB in F Statistical significance was assessed using Student's t-test in F, significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.


Figure 5A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment ( black and red ) and upon treatment with adenylyl cyclase activator forskolin ( gray and pink ) or PKA inhibitor H89 ( light grey and orange ) . Data information : The scale bar is 2 µm in AC , D . Quantification of the RRP and TRP fraction from experiment in A and B . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in ( C and D ) , The statistical significance was assessed in C , D using two - way ANOVA with Tukey ´ s multiple comparison test * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E . Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA - dependent regulation of pSer9Syn1 . F , G . Quantification of pSer9Syn1 / Syn1 ratio ( in F ) and Syn1 ( in G ) on blots from E . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G , The statistical significance was assessed in F and G using Student ' s t - test as is depicted in graphs as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . H . Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1 . Neurons were pre - treated ( 4 µM , 1hr ) with KN93 to isolate PKA - dependent phosphorylation and then treated in addition with forskolin or H89 before fixation . I . Quantification of staining in H . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I . The statistical significance was assessed in I using two - way ANOVA with Tukey ´ s multiple comparison test as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The scale bar is 5 µm in H .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O

Figure 5A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (gray and pink) or PKA inhibitor H89 (light grey and orange). Data information: The scale bar is 2 µm in AC, D. Quantification of the RRP and TRP fraction from experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), The statistical significance was assessed in C, D using two-way ANOVA with Tukey´s multiple comparison test *p≤0.05, **p < 0.01, ***p< 0.001.E. Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA-dependent regulation of pSer9Syn1. F, G. Quantification of pSer9Syn1/Syn1 ratio (in F) and Syn1 (in G) on blots from E. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G, The statistical significance was assessed in F and G using Student's t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001.H. Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1. Neurons were pre-treated (4 µM, 1hr) with KN93 to isolate PKA-dependent phosphorylation and then treated in addition with forskolin or H89 before fixation. I. Quantification of staining in H. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I. The statistical significance was assessed in I using two-way ANOVA with Tukey´s multiple comparison test as *p≤0.05, **p < 0.01, ***p< 0.001. The scale bar is 5 µm in H.


Figure 6A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted for untreated WT and BsnGT neurons ( black and red ) and cultures treated with of 6 - Bnz - cAMPS ( 50 µM , 1 hour ; gray and pink ) and rolipram ( 1 µM , 30 min , light grey and orange ) . Data information : Scale bar is 2µm . C , D . Quantifications of RRP and TRP fractions in experiment in A and B . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison test in C , D : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . H . Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student ' s t - test in H and is given as : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . I . Representative immunoblot with antibody against CDK5 - dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals .

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 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 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 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

Figure 6A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for untreated WT and BsnGT neurons (black and red) and cultures treated with of 6-Bnz-cAMPS (50 µM, 1 hour; gray and pink) and rolipram (1 µM, 30 min, light grey and orange). Data information: Scale bar is 2µm.C, D. Quantifications of RRP and TRP fractions in experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test in C, D : *p≤0.05, **p < 0.01, ***p< 0.001.H. Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student's t-test in H and is given as: *p≤0.05, **p < 0.01, ***p< 0.001.I. Representative immunoblot with antibody against CDK5-dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals.


Figure 1Relative quantification of mRNA for Ppcs , Ppcdc , and Coasy by genotype from each of the three study regions . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively ) Western blot and quantification for Coasy from GP by genotype and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . Ponceau S was used as a loading control . n = 8 , 4 for WT and n = 8 , 6 for KO ( vehicle and pPanSH groups , respectively ) .

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Figure 1Relative quantification of mRNA for Ppcs, Ppcdc, and Coasy by genotype from each of the three study regions. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)Western blot and quantification for Coasy from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as a loading control. n=8, 4 for WT and n=8, 6 for KO (vehicle and pPanSH groups, respectively).


Figure 2Relative mRNA expression of Tfrc , Ireb2 , and Hamp by genotype and brain region . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively ) Total iron quantity in cytosol and mitochondria from GP using ICP - MS . The data distribution is presented using a box plot , including minimum , first quartile , median , third quartile , and maximum . The magenta - colored line represent mean . n = 6 for both genotypesAssay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype . Quantification of aconitase activity in two tissues ( brain and liver ) from animals of each genotype by band density . n = 4 for WT , n = 5 for KO ( D - E ) Activity of pyruvate dehydrogenaseAssay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype . Quantification of aconitase activity in two tissues ( brain and liver ) from animals of each genotype by band density . n = 4 for WT , n = 5 for KO ( D - E ) Activity of pyruvate dehydrogenase ( D ) and complex I ( E ) in GP by genotype and treatment status ( veh = vehicle ; pPanSH = 4 ' - phosphopantetheine ) . n = 5 for both genotypes and treatment groups . Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control .

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Figure 2Relative mRNA expression of Tfrc, Ireb2, and Hamp by genotype and brain region. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)Total iron quantity in cytosol and mitochondria from GP using ICP-MS. The data distribution is presented using a box plot, including minimum, first quartile, median, third quartile, and maximum. The magenta-colored line represent mean. n=6 for both genotypesAssay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenaseAssay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase (D) and complex I (E) in GP by genotype and treatment status (veh = vehicle; pPanSH = 4'-phosphopantetheine). n=5 for both genotypes and treatment groups.Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control.


Figure 3Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype , brain region and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively ) QRT - PCR . n = 5 for Western blot for all groupsRelative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region . n = same as 3A . Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype . n = 5 for WT and n = 3 for KO .

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Figure 3Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype, brain region and treatment status (pPanSH = 4'-phosphopantetheine). n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) QRT-PCR. n=5 for Western blot for all groupsRelative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region. n= same as 3A.Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype. n=5 for WT and n=3 for KO.


Figure 4Relative quantification of COASY mRNA in human PKAN primary cell lines . n = 5 for both genotypes and cell typesRelative quantification of COASY / Coasy in fresh , blood - derived lymphocytes from human and mouse compared by genotype . n = 51 , 35 for human ( control , PKAN , respectively ) and n = 5 for both mouse genotypes . Extracellular flux analysis in human fibroblasts showing differences in OCR ( oxygen consumption rate ) and ECAR ( extracellular acidification rate ) by genotype . Oligom ; Oligomycin , FCCP ; Carbonyl cyanide - ptrifluoromethoxyphenylhydrazone , 2 - DG ; 2 - deoxy - glucose . Quantification of baseline OCR and ECAR is shown , as well . n = 3 per genotype with four technical replicates . Complex I activity assay using fibroblast lysates . Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n = 4 for both genotypes and treatment groups . Relative mRNA expression of mitochondrially - encoded genes important for oxidative phosphorylation from complex I ( MT - ND1 ) , complex III ( MT - CYB ) , cytochrome C ( MT - CO1 ) , and ATP synthase ( MT - ATP6 ) in human mutant fibroblasts versus control cells . n = 3 for both genotypes . Calculation of active mitochondria using the JC - 1 assay by genotype in control and PKAN lymphoblasts .

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

Figure 4Relative quantification of COASY mRNA in human PKAN primary cell lines. n= 5 for both genotypes and cell typesRelative quantification of COASY/Coasy in fresh, blood-derived lymphocytes from human and mouse compared by genotype. n=51,35 for human (control, PKAN, respectively) and n=5 for both mouse genotypes.Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; Oligomycin, FCCP; Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy-glucose. Quantification of baseline OCR and ECAR is shown, as well. n=3 per genotype with four technical replicates.Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n=4 for both genotypes and treatment groups.Relative mRNA expression of mitochondrially-encoded genes important for oxidative phosphorylation from complex I (MT-ND1), complex III (MT-CYB), cytochrome C (MT-CO1), and ATP synthase (MT-ATP6) in human mutant fibroblasts versus control cells. n=3 for both genotypes.Calculation of active mitochondria using the JC-1 assay by genotype in control and PKAN lymphoblasts.


Figure 5Relative expression of Pank1α , Pank1β , total Pank1 and Pank3 in mouse brain regions . n = 8 , 8 , 4 for both genotypes ( GP , SN , Cerebellum , respectively ) . Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate ( 0 . 8 mg / g body weight ) or gemfibrozil ( 1 . 2 mg / g body weight ) . n = 8 for both genotypes treated with vehicle , n = 5 for both KO groups treated with either bezafibrate or gemfibrozil .

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Figure 5Relative expression of Pank1α, Pank1β, total Pank1 and Pank3 in mouse brain regions. n=8,8,4 for both genotypes (GP, SN, Cerebellum, respectively).Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate (0.8 mg/g body weight) or gemfibrozil (1.2 mg/g body weight). n=8 for both genotypes treated with vehicle, n=5 for both KO groups treated with either bezafibrate or gemfibrozil.


Figure 6Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds : 4 ' - phosphopantetheine ( pPanSH ) , pantetheine , vitamin B5 , and CoA . n = 8 ( WT - veh ) , 5 ( KO - veh ) , 5 ( KO - pPanSH ) , 4 ( KO - all other intermediates ) . Correction of Coasy , Tfrc , Ireb2 , and Drd1 expression in GP by 4 ' - phosphopantetheine over a range of doses . n = 8 ( WT - veh ) , 5 ( KO - veh ) , 5 ( KO - 0 . 82 ) , 4 ( WT - 5 ) , 5 ( KO - 5 ) , 5 ( KO - 8 . 2 ) , 5 ( KO - 10 ) . 5 ( WT - 20 ) , 5 ( KO - 20 ) Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH . n = 3 for both genotypes . ECAR measured in cultured human fibroblasts by genotype and treatment status . Cells were treated for three days with 4 ' - phosphopantetheine ( pPanSH ) or vehicle ( veh ) . n = 3 for both genotypes .

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Figure 6Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds: 4'-phosphopantetheine (pPanSH), pantetheine, vitamin B5, and CoA. n=8 (WT-veh), 5 (KO-veh), 5 (KO-pPanSH), 4 (KO-all other intermediates). Correction of Coasy, Tfrc, Ireb2, and Drd1 expression in GP by 4'-phosphopantetheine over a range of doses. n=8 (WT-veh), 5 (KO-veh), 5 (KO-0.82), 4 (WT-5), 5 (KO-5), 5 (KO-8.2), 5 (KO-10). 5 (WT-20), 5 (KO-20)Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH. n=3 for both genotypes.ECAR measured in cultured human fibroblasts by genotype and treatment status. Cells were treated for three days with 4'-phosphopantetheine (pPanSH) or vehicle (veh). n=3 for both genotypes.


Figure 7Relative quantification of Coasy , Tfrc and Drd1 mRNA from GP by number of days post - cessation of treatment with 5μg of 4 ' - phosphopantetheine per g of body weight . n = 11 ( WT - veh - D0 ) , 5 ( KO - veh - D0 ) , 4 ( WT - pPanSH - D0 ) , 5 ( KO - pPanSH - D0 ) , 5 ( KO - pPanSH - D1 ) , 5 ( KO - pPanSH - D2 ) , 5 ( KO - pPanSH - D3 ) , 5 ( WT - pPanSH - D7 ) , 4 ( KO - pPanSH - D7 ) ,

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Figure 7Relative quantification of Coasy, Tfrc and Drd1 mRNA from GP by number of days post-cessation of treatment with 5μg of 4'-phosphopantetheine per g of body weight. n= 11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-pPanSH-D7), 4 (KO-pPanSH-D7),


Figure 1Biological replicates of transcriptional profiles ( Tx ) measured in separately prepared cell free lysate batches of E . coli . Correlation between transcriptional profiles ( Tx ) for regulatory sequence libraries from in vitro and in vivo measurements in E . coli . The color scale indicates the density of data points in a given area of the plot . Correlation between primary TSS calls ( in bp from ATG start codon ) of regulatory sequences from in vitro and in vivo measurements in E . coli .

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Figure 1Biological replicates of transcriptional profiles (Tx) measured in separately prepared cell free lysate batches of E. coli.Correlation between transcriptional profiles (Tx) for regulatory sequence libraries from in vitro and in vivo measurements in E. coli. The color scale indicates the density of data points in a given area of the plot.Correlation between primary TSS calls (in bp from ATG start codon) of regulatory sequences from in vitro and in vivo measurements in E. coli.


Figure 2Optimization of transcriptional output using different concentrations of Mg - glutamate and K - glutamate in 10 bacterial cell - free expression systems with Broccoli as a reporter ( solid lines , optimal buffer composition shown in red ) . No DNA template controls are shown as grey dashed lines . 12 . 5 nM of DNA template was used in all systems , except L . lactis ( 50 nM used ) .

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Figure 2Optimization of transcriptional output using different concentrations of Mg-glutamate and K-glutamate in 10 bacterial cell-free expression systems with Broccoli as a reporter (solid lines, optimal buffer composition shown in red). No DNA template controls are shown as grey dashed lines. 12.5 nM of DNA template was used in all systems, except L. lactis (50 nM used).


Figure 3Transcriptional activities ( Tx ) of 421 regulatory sequences that were active in all 10 bacterial cell - free expression systems . Pairwise comparison of transcriptional profiles of 421 universally - active regulatory sequences between bacterial species . Example scatter plots with relatively high and low Pearson correlation in the heatmap ( marked i and ii ) are shown on left . Correlation between evolutionary divergences ( 16S rRNA percent identity ) and pairwise Pearson correlation of transcriptional profiles . Dashed line represents linear regression .

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 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 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

Figure 3Transcriptional activities (Tx) of 421 regulatory sequences that were active in all 10 bacterial cell-free expression systems.Pairwise comparison of transcriptional profiles of 421 universally-active regulatory sequences between bacterial species. Example scatter plots with relatively high and low Pearson correlation in the heatmap (marked i and ii) are shown on left.Correlation between evolutionary divergences (16S rRNA percent identity) and pairwise Pearson correlation of transcriptional profiles. Dashed line represents linear regression.


Figure 5Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for ( d ) E . coli + B . subtilisPairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for ( e ) E . coli + C . glutamicum hybrids with different mixing ratios .

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 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

Figure 5Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (d) E. coli + B. subtilisPairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (e) E. coli + C. glutamicum hybrids with different mixing ratios.


Figure 1A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young and old mice , respectively . B : WT1 immunohistochemistry ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young and old mice , respectively . C : TUNEL assay ( upper panels ) and quantification of TUNEL - positive tubular cells ( upper panel ) in glomeruli from young and aged mice . Panels are representative images of 6 young and old mice , respectively . Scale bar = 20 μm . D : Senescence - associated β - galactosidase staining ( upper panels ) and quantification of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . Panels are representative images of 4 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice . E : p21 / griffonia simplicifolia / nephrin coimmunostaining in kidneys from young and aged mice ( left panels ) and quantification ( right panel ) of p21 - positive endothelial cells per glomeruli . Original magnification X630 . Scale bar = 10 μm . Panels are representative images of 6 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice . F : 53BP1 / CD34 co - immunostaining in kidneys from young and aged mice ( left panels ) and quantification ( right panels ) of 53BP1 foci per glomerular endothelial cell . Original magnification X630 . Scale bar = 10 μm . Panels are representative images of 6 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice . I : PAI - 1 immunohistochemistry ( left panels ) and quantification of PAI - 1 - positive glomeruli ( right panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 5 for young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 1A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive tubular cells (upper panel) in glomeruli from young and aged mice. Panels are representative images of 6 young and old mice, respectively. Scale bar = 20 μm. D: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. Panels are representative images of 4 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.E: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys from young and aged mice (left panels) and quantification (right panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.F: 53BP1/CD34 co-immunostaining in kidneys from young and aged mice (left panels) and quantification (right panels) of 53BP1 foci per glomerular endothelial cell. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.I: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 5 for young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


Figure 2A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 4 and n = 5 for RT + and RT - , respectively . B : WT1 immunohistochemistry ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + and RT - mice , respectively . C : Senescence - associated β - galactosidase staining ( upper panels ) and quantification of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice . E : p21 / griffonia simplicifolia / nephrin coimmunostaining ( left panels ) and quantification of endothelial p21 - positive cells ( right panel ) in glomeruli of kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice . F : 53BP1 / CD34 coimmunostaining ( left panels ) and quantification of glomerular endothelial cell 53BP1 foci ( right panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + and RT - mice , respectively . Arrows show 53BP1 foci . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice . H : PAI - 1 immunohistochemistry ( left panels ) and quantification of PAI - 1 - positive glomeruli ( right panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . n = 4 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 2A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 5 for RT+ and RT-, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. C: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.E: p21/griffonia simplicifolia/nephrin coimmunostaining (left panels) and quantification of endothelial p21-positive cells (right panel) in glomeruli of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.F: 53BP1/CD34 coimmunostaining (left panels) and quantification of glomerular endothelial cell 53BP1 foci (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Arrows show 53BP1 foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.H: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


Figure 3A : WT1 ( left upper panels ) and p21 ( left middle and lower panels ) immunostaining in 4 - , 12 - , 18 - and 24 - month - old mice . Quantification of WT1 - positive cells ( right upper panels ) , and glomerular and tubular p21 - postive cells ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . B : WT1 ( left upper panels ) and p21 ( left middle and lower panels ) immunostaining in 4 - and 12 - month - old irradiated ( RT + ) and non - irradiated ( RT - ) mice . Quantification of WT1 - positive cells ( right upper panels ) , and glomerular and tubular p21 - postive cells ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4-, 12-, 18- and 24-month-old mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. B: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4- and 12-month-old irradiated (RT+) and non-irradiated (RT-) mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test.


Figure 4A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . B : WT1 immunostaining ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . C : PAI - 1 immunostaining ( upper panels ) and quantification of PAI - 1 - positive glomeruli ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . Data information : Data are means ± SEM . Statistical analysis : t - student test : AP + versus AP - mice .

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 O O O O O O O O O O O O O O O O O O O O O O O O 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 4A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. B: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. C: PAI-1 immunostaining (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. Data information: Data are means ± SEM. Statistical analysis: t-student test: AP+ versus AP- mice.


Figure 5A : PAI - 1 immunohistochemistry ( upper panels ) and quantification of PAI - 1 - positive glomeruli ( lower panel ) in kidneys of PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 4 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . B : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 12 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . C : TUNEL assay ( upper panels ) and quantification of TUNEL - positive glomeruli ( lower panel ) in kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . D : WT1 immunostaining ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : PAI - 1 ∆ endo versus PAI - 1flox mice . G : p21 / griffonia simplicifolia / nephrin coimmunostaining in kidneys ( upper panels ) from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age and quantification ( lower panel ) of p21 - positive endothelial cells per glomeruli . Original magnification X630 . Scale bar = 10 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . H : 53BP1 / CD34 coimmunostaining ( upper panels ) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X630 . Scale bar = 10 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . Arrows show 53BP1 positive foci . Data information : Data are means ± SEM . Statistical analysis : t - student test : PAI - 1 ∆ endo versus PAI - 1flox mice .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O

Figure 5A: PAI-1 immunohistochemistry (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in kidneys of PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively. B: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 12 for PAI-1flox and PAI-1∆endo mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. D: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.G: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys (upper panels) from PAI-1flox and PAI-1∆endo mice at 22 months of age and quantification (lower panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. H: 53BP1/CD34 coimmunostaining (upper panels) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X630. Scale bar = 10 μm. n=4 and n=6 for PAI-1flox and PAI-1∆endo mice, respectively. Arrows show 53BP1 positive foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.


Figure 6C : Relative mRNA expression of senescence markers ( p16 and p21 ) and SASP molecules ( PAI - 1 , IL6 , IL8 ) in GEnC at early ( p9 ) or late ( p17 ) passage . n = 5 independent experiments . Data information : Data are means ± SEM . Statistical analysis : t - student testE : Immunofluorescence of paxilin ( green ) and phalloidin ( red ) in podocytes stimulated with control medium , supernatant ( SN ) from glomerular endothelial cells from early ( p9 ) or late ( p17 ) passage and treated either with vehicle or Tiplaxtinin , a PAI - 1 inhibitor ( upper panel ) . Quantification of focal adhesions ( lower panel ) was performed by counting the number of paxilin - positive spots in 30 cells from three independent experiments . Original magnification X1000 . Scale bar = 20 μm . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer

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 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

Figure 6C: Relative mRNA expression of senescence markers (p16 and p21) and SASP molecules (PAI-1, IL6, IL8) in GEnC at early (p9) or late (p17) passage. n = 5 independent experiments. Data information: Data are means ± SEM. Statistical analysis: t-student testE: Immunofluorescence of paxilin (green) and phalloidin (red) in podocytes stimulated with control medium, supernatant (SN) from glomerular endothelial cells from early (p9) or late (p17) passage and treated either with vehicle or Tiplaxtinin, a PAI-1 inhibitor (upper panel). Quantification of focal adhesions (lower panel) was performed by counting the number of paxilin-positive spots in 30 cells from three independent experiments. Original magnification X1000. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test


Figure 7A : PAI - 1 staining ( left panel ) and quantification ( right panel ) in glomeruli of kidney biopsies from young ( < 40 years of age , n = 8 ) or old ( > 80 years of age , n = 10 ) kidney donors at time of transplantation ( M0 ) . Original magnification X400 . Scale bar = 20 μm . B : p16 staining ( left panel ) and quantification ( right panel ) in glomeruli of kidney biopsies from young ( < 40 years of age , n = 8 ) or old ( > 80 years of age , n = 10 ) kidney donors at time of transplantation ( M0 ) . The arrows point to the staining . Original magnification X400 . Scale bar = 20 μm . Data information : Data are means ± SEM . Statistical analysis : t - student testF : Quantification of the glomerulosclerosis score at time of ( M0 ) , or 12 months after ( M12 ) kidney transplantation from elderly donors in patients displaying a negative ( PAI - 1 - , n = 17 ) or positive ( PAI - 1 + , n = 13 ) glomerular PAI - 1 staining at time of transplantation ( M0 ) . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer testH : Urinary PAI - 1 measured by ELISA in a cohort of elderly patients with ( n = 48 ) or without ( n = 48 ) chronic kidney disease . Data information : Data are means ± SEM . Statistical analysis : t - student

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 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 O

Figure 7A: PAI-1 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). Original magnification X400. Scale bar = 20 μm. B: p16 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). The arrows point to the staining. Original magnification X400. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: t-student testF: Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12) kidney transplantation from elderly donors in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 13) glomerular PAI-1 staining at time of transplantation (M0). Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer testH: Urinary PAI-1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease. Data information: Data are means ± SEM. Statistical analysis: t-student test


Figure 1 ( a ) Representative micro - CT pictures of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Note the severe osteoporosis in A20myel - KO mice . ( b ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Each dot represents an individual mouse ( A20WT , n = 10 ; A20myel - KO , n = 8 ) .

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Figure 1(a) Representative micro-CT pictures of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Note the severe osteoporosis in A20myel-KO mice. (b) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Each dot represents an individual mouse (A20WT, n=10; A20myel-KO, n=8).


Figure 2 ( a ) Levels of TNF and IL - 6 in serum of A20WT , A20OC - KO and A20myel - KO mice . Each dot represents an individual mouse ( A20WT , n = 11 ; A20OC - KO , n = 11 and A20myel - KO , n = 14 ) . Data are expressed as mean ± SEM . * ( c ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20OC - KO littermates . Each dot represents an individual mouse ( A20WT , n = 6 ; A20OC - KO , n = 9 ) . ( e ) Representative pictures of TRAP - stained sections from tibia of 20 week - old A20WT and A20OC - KO mice . Red arrows indicate osteoclasts . ( f ) Quantification of the number of TRAP - positive cells on sections from tibia of 20 week - old A20WT and A20OC - KO mice . Each dot represents an individual mouse ( A20WT , n = 5 ; A20OC - KO , n = 6 ) .

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 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 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 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

Figure 2(a) Levels of TNF and IL-6 in serum of A20WT, A20OC-KO and A20myel-KO mice. Each dot represents an individual mouse (A20WT, n=11; A20OC-KO, n=11 and A20myel-KO, n=14). Data are expressed as mean ± SEM. *(c) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20OC-KO littermates. Each dot represents an individual mouse (A20WT, n=6; A20OC-KO, n=9).(e) Representative pictures of TRAP-stained sections from tibia of 20 week-old A20WT and A20OC-KO mice. Red arrows indicate osteoclasts.(f) Quantification of the number of TRAP-positive cells on sections from tibia of 20 week-old A20WT and A20OC-KO mice. Each dot represents an individual mouse (A20WT, n=5; A20OC-KO, n=6).


Figure 3 ( d ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF - κB reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and with either an empty vector or a vector expressing wild - type A20 . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency . ( e ) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel - KO mice and stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control . ( f ) Wild - type and A20 deficient BMDMs were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for RANK , A20 , TRAF6 , HOIP and Sharpin . ( g ) BMDMs from A20WT and A20myel - KO mice were stimulated with RANKL ( 100ng / ml ) for the indicated time periods and immunoblotted to visualize p100 processing to p52 . Actin is shown as a loading control .

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 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 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 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

Figure 3(d) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF-κB reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and with either an empty vector or a vector expressing wild-type A20. Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency.(e) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel-KO mice and stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.(f) Wild-type and A20 deficient BMDMs were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for RANK, A20, TRAF6, HOIP and Sharpin.(g) BMDMs from A20WT and A20myel-KO mice were stimulated with RANKL (100ng/ml) for the indicated time periods and immunoblotted to visualize p100 processing to p52. Actin is shown as a loading control.


Figure 4 ( a ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transfected with an NF - κB luciferase reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and plasmids encoding different A20 variants : A20WT , A20DUB ( C103A mutation ) , A20ZnF4 ( C624A / C627A ) , A20ZnF7 ( C775A / C779A ) , and A20ZnF4ZnF7 ( C624A / C627A / C775A / C779A ) . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency . Data are expressed as the mean of technical triplicates ± SD . ( c - d ) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice , stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control . ( e ) BMDMs isolated from control ( A20WT ) , A20ZnF7 , A20ZnF4ZnF7 and A20DUB mice were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control . ( f ) Wild - type BMDMs were pretreated for 1h with HOIPIN - 8 ( 50µM ) or left untreated and stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control .

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 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 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 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

Figure 4(a) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transfected with an NF-κB luciferase reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and plasmids encoding different A20 variants: A20WT, A20DUB (C103A mutation), A20ZnF4 (C624A/C627A), A20ZnF7 (C775A/C779A), and A20ZnF4ZnF7 (C624A/C627A/C775A/C779A). Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency. Data are expressed as the mean of technical triplicates ± SD.(c-d) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice, stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.(e) BMDMs isolated from control (A20WT), A20ZnF7, A20ZnF4ZnF7 and A20DUB mice were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.(f) Wild-type BMDMs were pretreated for 1h with HOIPIN-8 (50µM) or left untreated and stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.


Figure 1 ( B ) Proteasome inhibitor treatment ( 10 μM MG132 for 1 to 8 h ) promotes accumulation of a ~ 110KD fragment ( asterisk ) in HEK293A cells . ( C ) The ~ 110KD fragment ( asterisk ) is related to AMOT . Wild - type ( WT ) and AMOT knockout ( KO ) HEK293A cells were treated with 10 μM proteasome inhibitor bortezomib ( BTZ ) for 6 h and subjected to Western blotting analysis using the indicated antibodies . ( F and G ) Serum and LPA treatments induce AMOT degradation and promote accumulation of a ~ 110 kDa protein ( asterisk ) in HEK293A cells . Cells were serum - starved overnight and then treated with 10 % FBS or 1 μM LPA for the indicated time . The phosphorylation levels of YAP proteins were detected by Phos - tag gel and pYAP immunoblotting . Protein quantification is shown below . Data represent mean ± SEM from three independent experiments . L . E . indicates long exposure .

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 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 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 1(B) Proteasome inhibitor treatment (10 μM MG132 for 1 to 8 h) promotes accumulation of a ~110KD fragment (asterisk) in HEK293A cells.(C) The ~110KD fragment (asterisk) is related to AMOT. Wild-type (WT) and AMOT knockout (KO) HEK293A cells were treated with 10 μM proteasome inhibitor bortezomib (BTZ) for 6 h and subjected to Western blotting analysis using the indicated antibodies.(F and G) Serum and LPA treatments induce AMOT degradation and promote accumulation of a ~110 kDa protein (asterisk) in HEK293A cells. Cells were serum-starved overnight and then treated with 10% FBS or 1 μM LPA for the indicated time. The phosphorylation levels of YAP proteins were detected by Phos-tag gel and pYAP immunoblotting. Protein quantification is shown below. Data represent mean ± SEM from three independent experiments. L.E. indicates long exposure.


Figure 2C ) Nelfinavir ( NFV ) and Lopinavir ( LPV ) block AMOT cleavage . HEK293A cells were pre - treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h . Cell lysates were then subjected to Western blotting . ( F ) Deletion of DDI2 blocks AMOT cleavage . Wild - type ( WT ) and DDI2 KO cells were serum - starved overnight and then treated with 1 μM LPA for 2 h . ( G ) Both DDI1 and DDI2 mediate AMOT cleavage . Ectopically expressed FLAG - tagged DDI1 or DDI2 rescues AMOT cleavage in DDI2 KO HEK293A cells . K ) The aspartic catalytic - site mutant DDI2 exhibits a dominant negative effect through inactivation of wild - type DDI2 in HEK293A cells . Wild - type HEK293A cells were transfected with the indicated plasmids and treated with 1 μM LPA ( K ) before harvest .

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Figure 2C) Nelfinavir (NFV) and Lopinavir (LPV) block AMOT cleavage. HEK293A cells were pre-treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h. Cell lysates were then subjected to Western blotting.(F) Deletion of DDI2 blocks AMOT cleavage. Wild-type (WT) and DDI2 KO cells were serum-starved overnight and then treated with 1 μM LPA for 2 h.(G) Both DDI1 and DDI2 mediate AMOT cleavage. Ectopically expressed FLAG-tagged DDI1 or DDI2 rescues AMOT cleavage in DDI2 KO HEK293A cells.K) The aspartic catalytic-site mutant DDI2 exhibits a dominant negative effect through inactivation of wild-type DDI2 in HEK293A cells. Wild-type HEK293A cells were transfected with the indicated plasmids and treated with 1 μM LPA (K) before harvest.


Figure 3 ( A ) Ubiquitination is required for AMOT cleavage . HEK293A cells were treated with 100 nM E1 inhibitor MLN7243 overnight followed by 10 μM BTZ for 6 h . Cell lysates were then subjected to Western blotting . ( B and C ) The TNKS1 / 2 - RNF146 axis is crucial for AMOT cleavage . Wild - type ( WT ) , TNKS1 / 2 double KO ( dKO ) and RNF146 KO HEK293A cells were treated with the proteasome inhibitor MG132 for 6 h before harvest . Cell lysates were subjected to Western blotting analysis using the indicated antibodies . ( H and I ) AMOT cleavage states in different cell lines with NF2 - wild - type or NF2 - deficiency . ( K ) Serum , LPA , and S1P stimulate PARylation and ubiquitination of AMOT . Serum - starved cells were treated with 10 % FBS , 1 μM LPA , or 1 μM S1P for 4 h and subjected to an immunoprecipitation ( IP ) assay .

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Figure 3(A) Ubiquitination is required for AMOT cleavage. HEK293A cells were treated with 100 nM E1 inhibitor MLN7243 overnight followed by 10 μM BTZ for 6 h. Cell lysates were then subjected to Western blotting.(B and C) The TNKS1/2-RNF146 axis is crucial for AMOT cleavage. Wild-type (WT), TNKS1/2 double KO (dKO) and RNF146 KO HEK293A cells were treated with the proteasome inhibitor MG132 for 6 h before harvest. Cell lysates were subjected to Western blotting analysis using the indicated antibodies.(H and I) AMOT cleavage states in different cell lines with NF2-wild-type or NF2-deficiency.(K) Serum, LPA, and S1P stimulate PARylation and ubiquitination of AMOT. Serum-starved cells were treated with 10% FBS, 1 μM LPA, or 1 μM S1P for 4 h and subjected to an immunoprecipitation (IP) assay.


Figure 4 ( C ) Nelfinavir and Lopinavir block AMOT cleavage in HUVECs . HUVECs were pre - treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h . Cell lysates were subjected to Western blotting . Protein quantification is shown below . ( D ) Aspartic protease DDI2 is responsible for AMOT cleavage in HUVECs . HUVECs were transfected with two different small interfering RNA ( siRNA ) and 48 h post - transfection , the cells were treated with 10 μM BTZ for 6 h . Cell lysates were subjected to Western blotting . Protein quantification is shown below . ( F RNF146 are indispensable for AMOT cleavage in HUVECs . siRNAs were transfected into HUVECs , the cells were treated with 10 μM BTZ for 6 h before harvest . Protein quantifications are shown below . ( H ) Representative immunofluorescence images of HUVEC spheroids 18 h after VEGF stimulation . Above : inverted contrast images . Areas within the red dash box indicate region of interest ( ROI ) and are shown below at higher magnification . Plasma membranes were stained with CellMask ( red ) , actin filaments with Phalloidin ( green ) , and nucleus with DAPI ( blue ) . Scale bar 100 μm . HUVECs infected with empty vector virus were used as control . ( I and J ) Quantification of the number of sprouts per spheroid and the cumulative sprout length of HUVECs . Vector ( n = 20 ) , AMOT - WT ( n = 18 ) , AMOT - ΔTBD ( n = 14 ) , AMOT - Δcut ( n = 15 ) , AMOTp80 ( n = 16 ) , AMOT - NT ( n = 17 ) and AMOT - CT ( n = 21 ) .

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 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 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 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

Figure 4(C) Nelfinavir and Lopinavir block AMOT cleavage in HUVECs. HUVECs were pre-treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h. Cell lysates were subjected to Western blotting. Protein quantification is shown below. (D) Aspartic protease DDI2 is responsible for AMOT cleavage in HUVECs. HUVECs were transfected with two different small interfering RNA (siRNA) and 48 h post-transfection, the cells were treated with 10 μM BTZ for 6 h. Cell lysates were subjected to Western blotting. Protein quantification is shown below.(F RNF146 are indispensable for AMOT cleavage in HUVECs. siRNAs were transfected into HUVECs, the cells were treated with 10 μM BTZ for 6 h before harvest. Protein quantifications are shown below.(H) Representative immunofluorescence images of HUVEC spheroids 18 h after VEGF stimulation. Above: inverted contrast images. Areas within the red dash box indicate region of interest (ROI) and are shown below at higher magnification. Plasma membranes were stained with CellMask (red), actin filaments with Phalloidin (green), and nucleus with DAPI (blue). Scale bar 100 μm. HUVECs infected with empty vector virus were used as control.(I and J) Quantification of the number of sprouts per spheroid and the cumulative sprout length of HUVECs. Vector (n=20), AMOT-WT (n=18), AMOT-ΔTBD (n=14), AMOT-Δcut (n=15), AMOTp80 (n=16), AMOT-NT (n=17) and AMOT-CT (n=21).


Figure 5 ( A and B ) Knockdown of AMOT induces vascular development defects in zebrafish embryos . Lateral views of ISV and DLAV formation in Tg ( flk : GFP ) control and amot MO - injected zebrafish embryos at 30 h post - fertilization ( hpf ) are shown . Histogram depicts the quantification of normal and angiogenesis - defective embryos at 30 hpf ( B ) . n : number of embryos analyzed . Dorsolateral anastomose vessels ( DLAV ) , intersegmental vessels ( ISV ) , dorsal aorta ( DA ) and posterior cardinal vein ( PCV ) are shown . Asterisks : defective ISV ; dotted lines : dorsal edge of the embryo . Scale bar : 50 μm . ( E and F ) Non - cleavable AMOT mutant fails to support angiogenesis . Embryos were injected with amot MO together with mRNA encoding wild - type ( WT ) or mutant ( ΔTBD , NT , CT , and Δcut ) AMOT . Scale bar : 50 μm . ( G and H ) AMOT - CT but not full - length AMOT could rescue impaired angiogenesis in ddi2 - deficient embryos . Embryos were injected with ddi2 MO together with mRNA encoding wild - type AMOT ( AMOT - WT ) or AMOT - CT . Scale bar : 50 μm . ( I and J ) Nelfinavir ( NFV ) and XAV939 repress ISV development . Embryos were treated with 5 μM NFV , 10 μM XAV939 at 6 hpf for 24 h . Number of ISV defects was counted at 30 hpf . Scale bar : 50 μm .

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Figure 5(A and B) Knockdown of AMOT induces vascular development defects in zebrafish embryos. Lateral views of ISV and DLAV formation in Tg(flk:GFP) control and amot MO-injected zebrafish embryos at 30 h post-fertilization (hpf) are shown. Histogram depicts the quantification of normal and angiogenesis-defective embryos at 30 hpf (B). n: number of embryos analyzed. Dorsolateral anastomose vessels (DLAV), intersegmental vessels (ISV), dorsal aorta (DA) and posterior cardinal vein (PCV) are shown. Asterisks: defective ISV; dotted lines: dorsal edge of the embryo. Scale bar: 50 μm.(E and F) Non-cleavable AMOT mutant fails to support angiogenesis. Embryos were injected with amot MO together with mRNA encoding wild-type (WT) or mutant (ΔTBD, NT, CT, and Δcut) AMOT. Scale bar: 50 μm.(G and H) AMOT-CT but not full-length AMOT could rescue impaired angiogenesis in ddi2-deficient embryos. Embryos were injected with ddi2 MO together with mRNA encoding wild-type AMOT (AMOT-WT) or AMOT-CT. Scale bar: 50 μm. (I and J) Nelfinavir (NFV) and XAV939 repress ISV development. Embryos were treated with 5 μM NFV, 10 μM XAV939 at 6 hpf for 24 h. Number of ISV defects was counted at 30 hpf. Scale bar: 50 μm.


Figure 6 ( G ) High magnification confocal images of filopodia extension at the leading edge of the retinal vascular in control and ΔcutiEC - TG mice . Scale bar : 10 μm . ( H ) Quantification of the number and average length of filopodia in control and ΔcutiEC - TG P6 retinal sprouts . Each data point represents the average of three measurements from one retina , control ( n = 7 ) and ΔcutiEC - TG ( n = 7 ) . ( L ) Δcut KI mouse retinal angiogenesis shows sprouting defects in the sprouting front border . Immunofluorescent images of P7 whole - mount retinas stained for IB4 and ERG . White dash rectangles indicate regions of interest ( ROI ) are shown below . Arrow heads : blunt - ended tip ECs . Scale bar : 100 μm . ( M ) Quantification of the retinal vasculature of control and Δcut KI mice at P7 . Each data point represents the average of two or three measurements from one retina , control ( n = 8 ) and Δcut KI ( n = 8 ) . ( N ) High magnification confocal images of filopodia extension at the leading edge of the retinal vascular in control and Δcut KI mice . Tip cells are labeled with IB4 . Scale bar : 10 μm . ( O ) Quantification of the number and average length of filopodia in control and Δcut KI P7 retinal sprouts . Each data point represents the average of three measurements from one retina , control ( n = 8 ) and Δcut KI ( n = 8 ) .

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 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 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

Figure 6(G) High magnification confocal images of filopodia extension at the leading edge of the retinal vascular in control and ΔcutiEC-TG mice. Scale bar: 10 μm. (H) Quantification of the number and average length of filopodia in control and ΔcutiEC-TG P6 retinal sprouts. Each data point represents the average of three measurements from one retina, control (n=7) and ΔcutiEC-TG (n=7).(L) Δcut KI mouse retinal angiogenesis shows sprouting defects in the sprouting front border. Immunofluorescent images of P7 whole-mount retinas stained for IB4 and ERG. White dash rectangles indicate regions of interest (ROI) are shown below. Arrow heads: blunt-ended tip ECs. Scale bar: 100 μm. (M) Quantification of the retinal vasculature of control and Δcut KI mice at P7. Each data point represents the average of two or three measurements from one retina, control (n=8) and Δcut KI (n=8).(N) High magnification confocal images of filopodia extension at the leading edge of the retinal vascular in control and Δcut KI mice. Tip cells are labeled with IB4. Scale bar: 10 μm. (O) Quantification of the number and average length of filopodia in control and Δcut KI P7 retinal sprouts. Each data point represents the average of three measurements from one retina, control (n=8) and Δcut KI (n=8).


Figure 7 ( D ) Ddi2iEC - KO mice show delayed retinal angiogenesis . Immunofluorescent images of whole - mount retinas stained for IB4 and ERG of control , Ddi2iEC - KO and Ddi2iEC - KO ; CTiEC - TG mice at P6 . White dash rectangles indicate regions of interest ( ROI ) are shown below at higher magnification . Scale bar : 100 μm . ( F ) High magnification confocal images of filopodia extension at the leading edge of the P6 retina . Tip cells are labeled with IB4 . Scale bar : 10 μm . ( G ) Quantification of the number and average length of filopodia in control ( n = 8 ) , Ddi2iEC - KO ( n = 9 ) and Ddi2iEC - KO ; CTiEC - TG ( n = 8 ) P6 retinal sprouts . Each data point represents the average of three measurements from one retina . ( H and I ) Representative images and comparisons of Ki67 positive endothelial cells in control ( n = 8 ) , Ddi2iEC - KO ( n = 8 ) and Ddi2iEC - KO ; CTiEC - TG ( n = 7 ) P6 retinas . Each data point represents the average of three measurements from one retina . Scale bar : 50 μm .

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 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 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

Figure 7(D) Ddi2iEC-KO mice show delayed retinal angiogenesis. Immunofluorescent images of whole-mount retinas stained for IB4 and ERG of control, Ddi2iEC-KO and Ddi2iEC-KO; CTiEC-TG mice at P6. White dash rectangles indicate regions of interest (ROI) are shown below at higher magnification. Scale bar: 100 μm.(F) High magnification confocal images of filopodia extension at the leading edge of the P6 retina. Tip cells are labeled with IB4. Scale bar: 10 μm. (G) Quantification of the number and average length of filopodia in control (n=8), Ddi2iEC-KO (n=9) and Ddi2iEC-KO; CTiEC-TG (n=8) P6 retinal sprouts. Each data point represents the average of three measurements from one retina.(H and I) Representative images and comparisons of Ki67 positive endothelial cells in control (n=8), Ddi2iEC-KO (n=8) and Ddi2iEC-KO; CTiEC-TG (n=7) P6 retinas. Each data point represents the average of three measurements from one retina. Scale bar: 50 μm.


Figure 8 ( D ) Nf2iEC - KO retinas show endothelial cell accumulation in the sprouting front and sparse vascular plexus in the region close to optic stalk . CT overexpression can partially rescue the phenotype . Immunofluorescent images of whole - mount P6 retinas stained for IB4 and ERG are shown . White rectangles indicate regions of interest ( ROI ) . Scale bar : 100 μm . ( F and G ) High magnification confocal images and quantification of filopodia extension at the leading edge of the P6 retinas . Tip cells are labeled with IB4 . Scale bar : 10 μm . Control ( n = 10 ) , Nf2iEC - KO ( n = 10 ) , Nf2iEC - KO ; CTiEC - TG ( n = 10 ) and Nf2iEC - KO ; ΔcutiEC - TG ( n = 8 ) . ( H and I ) Representative images and comparisons of Ki67 positive endothelial cells in control ( n = 8 ) , Nf2iEC - KO ( n = 8 ) , Nf2iEC - KO ; CTiEC - TG ( n = 8 ) and Nf2iEC - KO ; ΔcutiEC - TG ( n = 7 ) P6 retinas . Each data point represents the average of three measurements from one retina . Scale bar : 50 μm .

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 O O O O 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

Figure 8(D) Nf2iEC-KO retinas show endothelial cell accumulation in the sprouting front and sparse vascular plexus in the region close to optic stalk. CT overexpression can partially rescue the phenotype. Immunofluorescent images of whole-mount P6 retinas stained for IB4 and ERG are shown. White rectangles indicate regions of interest (ROI). Scale bar: 100 μm.(F and G) High magnification confocal images and quantification of filopodia extension at the leading edge of the P6 retinas. Tip cells are labeled with IB4. Scale bar: 10 μm. Control (n=10), Nf2iEC-KO (n=10), Nf2iEC-KO; CTiEC-TG (n=10) and Nf2iEC-KO; ΔcutiEC-TG (n=8).(H and I) Representative images and comparisons of Ki67 positive endothelial cells in control (n=8), Nf2iEC-KO (n=8), Nf2iEC-KO; CTiEC-TG (n=8) and Nf2iEC-KO; ΔcutiEC-TG (n=7) P6 retinas. Each data point represents the average of three measurements from one retina. Scale bar: 50 μm.


Figure 9 ( B ) CT overexpression promotes revascularization in the OIR model . Upper , IB4 staining of the whole retina of P15 control and CTiEC - TG mice after OIR . Lower , higher magnification images of sprouting vessels from veins . Scale bars are shown in the figures . ( D ) Blocking AMOT cleavage inhibits revascularization in the OIR model . Upper , IB4 staining of the whole retina of P17 WT and Δcut KI mice after OIR . Lower , higher magnification images of sprouting vessels from veins . Scale bars are shown in the figures .

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Figure 9(B) CT overexpression promotes revascularization in the OIR model. Upper, IB4 staining of the whole retina of P15 control and CTiEC-TG mice after OIR. Lower, higher magnification images of sprouting vessels from veins. Scale bars are shown in the figures.(D) Blocking AMOT cleavage inhibits revascularization in the OIR model. Upper, IB4 staining of the whole retina of P17 WT and Δcut KI mice after OIR. Lower, higher magnification images of sprouting vessels from veins. Scale bars are shown in the figures.


Figure 1A . Levels of ROW protein in rowRNAi lines were decreased in both male and female fly heads . Western blot for the two rowRNAi lines compared to controls was performed using rat polyclonal αROW and rat αTUBULIN ( TUB ) . Genotype description : rowRNAi - 1 ( Act - GAL4 / + ; UAS - rowRNAi - 1 / + ) , rowRNAi - 2 ( Act - GAL4 / + ; UAS - rowRNAi - 2 / + ) , rowRNAi - 1 Control ( CyO / + ; UAS - rowRNAi - 1 / + ) , rowRNAi - 2 Control ( CyO / + ; UAS - rowRNAi - 2 ) and Act - GAL4 Control ( Act - GAL4 / + ) . B . Decrease in the viability of rowRNAi lines . To examine the viability of rowRNAi flies , we counted the offspring generated from the cross between the heterozygous Act - GAL4 / CyO driver line with the homozygotes rowRNAi , or WT ( w1118 ) flies as control . Values are the percentages from the total progeny ± standard error of the mean ( SEM ) . C . Decrease in pupal eclosion of rowRNAi line . Pupae were collected from the cross between the heterozygous Act - GAL4 / CyO driver line with the homozygotes rowRNAi - 1 or WT ( w1118 ) flies as control . Values are the percentages for each genotype of pupal eclosion ± SEM . D . Survival curve for rowRNAi flies showing reduced life span compared to controls . n = 3 ( biological replicates ) . Significance was tested using the Kaplan - Meier estimate of survival and log - rank test . E . Crossing of females / males rowRNAi flies with WT males / females ( w1118 ) flies resulted in a strong reduction in offspring number . Values are the number of offspring from crosses between WT ( w1118 ) flies and flies with different genotypes .

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Figure 1A. Levels of ROW protein in rowRNAi lines were decreased in both male and female fly heads. Western blot for the two rowRNAi lines compared to controls was performed using rat polyclonal αROW and rat αTUBULIN (TUB). Genotype description: rowRNAi-1(Act-GAL4/+; UAS-rowRNAi-1/+), rowRNAi-2(Act-GAL4/+; UAS-rowRNAi-2/+), rowRNAi-1 Control (CyO/+; UAS-rowRNAi-1/+), rowRNAi-2 Control (CyO/+; UAS- rowRNAi-2) and Act-GAL4 Control (Act-GAL4 /+).B. Decrease in the viability of rowRNAi lines. To examine the viability of rowRNAi flies, we counted the offspring generated from the cross between the heterozygous Act-GAL4/CyO driver line with the homozygotes rowRNAi, or WT (w1118) flies as control. Values are the percentages from the total progeny ± standard error of the mean (SEM).C. Decrease in pupal eclosion of rowRNAi line. Pupae were collected from the cross between the heterozygous Act-GAL4/CyO driver line with the homozygotes rowRNAi-1 or WT (w1118) flies as control. Values are the percentages for each genotype of pupal eclosion ± SEM.D. Survival curve for rowRNAi flies showing reduced life span compared to controls. n=3 (biological replicates). Significance was tested using the Kaplan-Meier estimate of survival and log-rank test.E. Crossing of females/males rowRNAi flies with WT males/females (w1118) flies resulted in a strong reduction in offspring number. Values are the number of offspring from crosses between WT (w1118) flies and flies with different genotypes.


Figure 3A . Western blot for flies expressing endogenous FLAG - tagged ROW ( ROW - FLAG ) , with αFlag tag antibody to validate the tagging . αTubulin ( TUB ) antibody was used as a reference . Het , Heterozygotes ; Hom , Homozygotes for tagged ROW . D . BEAF - 32 coimmunoprecipitate with ROW . Lysates from S2 cells not transfected ( - ) or transfected ( + ) with ROW - FLAG tagged plasmid were subjected to immunoprecipitation with αFlag tag beads . The immunoprecipitates and input ( 5 % ) were analyzed by western blot with αFlag tag and αBEAF - 32 antibodies .

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Figure 3A. Western blot for flies expressing endogenous FLAG-tagged ROW (ROW-FLAG), with αFlag tag antibody to validate the tagging. αTubulin (TUB) antibody was used as a reference. Het, Heterozygotes; Hom, Homozygotes for tagged ROW.D. BEAF-32 coimmunoprecipitate with ROW. Lysates from S2 cells not transfected (-) or transfected (+) with ROW-FLAG tagged plasmid were subjected to immunoprecipitation with αFlag tag beads. The immunoprecipitates and input (5%) were analyzed by western blot with αFlag tag and αBEAF-32 antibodies.


Figure 5A . Central enrichment of AT - rich sequence in ROW binding regions . The logo shows the most significant enriched sequence ( based on MEME - ChIP analysis ) . The plot shows the motif ' s probability relative to the ROW ChIP - seq peaks ( calculated with centriMo ) . B . On the top are the results of DNA affinity pull - down followed by western blot with ROW antibody . None is a pull - down with no probe , AT - rich is a pull - down with a biotin - labeled AT - rich dsDNA probe , and control is a pull - down with a probe composed of 49 % A / T bases . On the bottom is the stain - free gel as a loading control . D . ChIP - qPCR results using FLAG - tag antibody for S2 cells transfected with WT or AT - hook mutant ROW - FLAG tagged plasmids ( n = 3 , technical and biological replicates ) . As a control , cells not transfected with ROW - FLAG tagged plasmid were used . Input percentages for 3 biological replicates are shown for three promoters containing AT - rich sequences ( Hcf , Phax , and Hcs ) . E . Co - IP results using αFlag antibody in S2 cells transfected with WT or AT - hook mutant ROW - FLAG tagged plasmids or with a plasmid containing only the ZNF domains of ROW ( with FLAG - tag ) . As a control , cells not transfected with ROW - FLAG tagged plasmid were used . Western blot using αFLAG , αWOC , αBEAF - 32 , αHP1c , and αTUB ( loading control ) antibodies is shown for input and IP samples .

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 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 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

Figure 5A. Central enrichment of AT-rich sequence in ROW binding regions. The logo shows the most significant enriched sequence (based on MEME-ChIP analysis). The plot shows the motif's probability relative to the ROW ChIP-seq peaks (calculated with centriMo).B. On the top are the results of DNA affinity pull-down followed by western blot with ROW antibody. None is a pull-down with no probe, AT-rich is a pull-down with a biotin-labeled AT-rich dsDNA probe, and control is a pull-down with a probe composed of 49% A/T bases. On the bottom is the stain-free gel as a loading control.D. ChIP-qPCR results using FLAG-tag antibody for S2 cells transfected with WT or AT-hook mutant ROW-FLAG tagged plasmids (n = 3, technical and biological replicates). As a control, cells not transfected with ROW-FLAG tagged plasmid were used. Input percentages for 3 biological replicates are shown for three promoters containing AT-rich sequences (Hcf, Phax, and Hcs).E. Co-IP results using αFlag antibody in S2 cells transfected with WT or AT-hook mutant ROW-FLAG tagged plasmids or with a plasmid containing only the ZNF domains of ROW (with FLAG-tag). As a control, cells not transfected with ROW-FLAG tagged plasmid were used. Western blot using αFLAG, αWOC, αBEAF-32, αHP1c, and αTUB (loading control) antibodies is shown for input and IP samples.