Figure 2 ( C ) Majority COUP - TFII + cells ( green ) were also positive for Foxd1 ( tdTomato - labeled as red ) in the non - injured mice kidney ( n = 2 ) . Quantification by confocal micrographs in 400x hpf .

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

Figure 2(C) Majority COUP-TFII+ cells (green) were also positive for Foxd1 (tdTomato-labeled as red) in the non-injured mice kidney (n=2). Quantification by confocal micrographs in 400x hpf.


Figure 3 ( A ) COUP - TFII expression is significantly increased and co - localized with αSMA + cells at day 10 of UUO and day 14 of UIRI in mice ( n = 3 animals per group ) . Quantification by confocal micrographs in 200x hpf . * p < 0 . 05 * * p < 0 . 01 by paired t test ; mean ± SD . ( B ) In a time course of UUO , COUP - TFII expression increased as early as day 2 and preceded the increased expression of αSMA ( n = 2 animals per time point ) . Quantification by confocal micrographs in 200x hpf . * p < 0 . 05 ; * * * p < 0 . 001 by two - way ANOVA test ; mean ± SD . ( E & F ) Gli1 - tdTomato + cells expanded primarily in the outer medullary region . In contrast , COUP - TFII + cells are distributed both in cortex and medulla . A subset of COUP - TFII + cells ( green ) overlap with genetically labeled Gli1 + pericyte / perivascular cells ( red ) in UUO injury model ( n = 3 animals per group ) . CTL : contralateral kidney . Quantification by confocal micrographs in 200x hpf .

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

Figure 3(A) COUP-TFII expression is significantly increased and co-localized with αSMA+ cells at day 10 of UUO and day 14 of UIRI in mice (n=3 animals per group). Quantification by confocal micrographs in 200x hpf. *p<0.05 **p<0.01 by paired t test; mean ± SD.(B) In a time course of UUO, COUP-TFII expression increased as early as day 2 and preceded the increased expression of αSMA (n=2 animals per time point). Quantification by confocal micrographs in 200x hpf. *p<0.05; ***p<0.001 by two-way ANOVA test; mean ± SD.(E&F) Gli1-tdTomato+ cells expanded primarily in the outer medullary region. In contrast, COUP-TFII+ cells are distributed both in cortex and medulla. A subset of COUP-TFII+ cells (green) overlap with genetically labeled Gli1+ pericyte/perivascular cells (red) in UUO injury model (n=3 animals per group). CTL: contralateral kidney. Quantification by confocal micrographs in 200x hpf.


Figure 4 ( D ) COUP - TFII + cells decrease significantly in the UUO kidney in TAM - treated homozygous ( F / F ; Cre / + ) mice ( KO group , n = 6 ) compared to wild - type littermates ( WT group , n = 4 ) . Expression of αSMA ( red ) and collagen1 ( yellow ) are also markedly reduced . Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group . Quantification by confocal micrographs in 200x hpf . * * p < 0 . 01 by unpaired t test , mean ± SD .

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

Figure 4(D) COUP-TFII+ cells decrease significantly in the UUO kidney in TAM-treated homozygous (F/F;Cre/+) mice (KO group, n=6) compared to wild-type littermates (WT group, n=4). Expression of αSMA (red) and collagen1 (yellow) are also markedly reduced. Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group. Quantification by confocal micrographs in 200x hpf. **p<0.01 by unpaired t test, mean ± SD.


Figure 5 ( D ) COUP - TFII - OE cells , in the absence of TGFβ1 stimulation , displayed an elongated fibroblast shape with increased αSMA expression , similar to WT cells treated with TGFβ1 . Quantification by confocal micrographs in 400x hpf , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA , mean ± SD . ( E ) Overexpression of COUP - TFII alone without TGFβ1 induces collagen1 production ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA , mean ± SD .

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 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

Figure 5(D) COUP-TFII-OE cells, in the absence of TGFβ1 stimulation, displayed an elongated fibroblast shape with increased αSMA expression, similar to WT cells treated with TGFβ1. Quantification by confocal micrographs in 400x hpf, *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.(E) Overexpression of COUP-TFII alone without TGFβ1 induces collagen1 production (n=3). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.


Figure 6 ( D ) WT , COUP - TFII overexpression ( OE ) and COUP - TFII knockout ( KO ) cells were seeded in Seahorse XF - 24 cell culture microplates . The cells were rendered quiescent in 0 . 5 % FBS DMEM overnight and then treated with or without 10 ng / ml TGFβ1 for 24 hours . All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate , followed by sequential treatments with glucose ( 10mM ) , oligomycin ( 5μg / ml ) and 2 - DG ( 50mM ) . Real - time extracellular acidification rate ( ECAR ) was recorded as the baseline ( before glucose ) , the rate of glycolysis ( after glucose ) , glycolytic capacity ( after oligomycin ) , and glycolytic reserve ( after 2 - DG ) ( n = 10 ) . ( E ) COUP - TFII OE significantly increased , while COUP - TFII KO decreased , in the baseline , rate of glycolysis , glycolytic capacity , and glycolytic reserve when compared to WT cells under TGFβ1 stimulation . Without TGFβ1 treatment , there is no difference on the baseline and glycolysis among WT , OE , and KO cells . COUP - TFII KO significantly decreased glycolytic capacity and reserve compared to WT , even without TGFβ1 stimulation ( n = 10 ) , * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

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 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

Figure 6(D) WT, COUP-TFII overexpression (OE) and COUP-TFII knockout (KO) cells were seeded in Seahorse XF-24 cell culture microplates. The cells were rendered quiescent in 0.5% FBS DMEM overnight and then treated with or without 10 ng/ml TGFβ1 for 24 hours. All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate, followed by sequential treatments with glucose (10mM), oligomycin (5μg/ml) and 2-DG (50mM). Real-time extracellular acidification rate (ECAR) was recorded as the baseline (before glucose), the rate of glycolysis (after glucose), glycolytic capacity (after oligomycin), and glycolytic reserve (after 2-DG) (n = 10).(E) COUP-TFII OE significantly increased, while COUP-TFII KO decreased, in the baseline, rate of glycolysis, glycolytic capacity, and glycolytic reserve when compared to WT cells under TGFβ1 stimulation. Without TGFβ1 treatment, there is no difference on the baseline and glycolysis among WT, OE, and KO cells. COUP-TFII KO significantly decreased glycolytic capacity and reserve compared to WT, even without TGFβ1 stimulation (n=10), *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.


Figure 7 ( A ) Volcano plot of all detected proteins from proteomics of TGFβ  - treated naïve ( WT ) and COUP - TFII overexpressing ( COUP - TFII - OE ) cells . PDK4 expression is significantly decreased in OE cells ( n = 2 ) . ( E ) PGC1α OE significantly increased expression of Cpt1 and PDK4 , both are involved in key steps of FAO . However , overexpression of PGC1α did not abrogate the TGFβ1 up - regulated genes in glycolysis and αSMA ( n = 6 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

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 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 7(A) Volcano plot of all detected proteins from proteomics of TGFβ-treated naïve (WT) and COUP-TFII overexpressing (COUP-TFII-OE) cells. PDK4 expression is significantly decreased in OE cells (n=2).(E) PGC1α OE significantly increased expression of Cpt1 and PDK4, both are involved in key steps of FAO. However, overexpression of PGC1α did not abrogate the TGFβ1 up-regulated genes in glycolysis and αSMA (n=6). *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.


Figure 1 ( B ) Non - supervised clustering heatmap plot indicates substantial clustering of transcripts from each tissue but not of transcripts from patients compared to controls . PD samples indicated in red . ( D ) qPCR validation of TH levels in SN samples of PD and controls , normalized to beta - actin mRNA . T - test p * = 0 . 025 , Data presented as mean ± SD . n = 18 for CT and 24 for PD .

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

Figure 1(B) Non-supervised clustering heatmap plot indicates substantial clustering of transcripts from each tissue but not of transcripts from patients compared to controls. PD samples indicated in red.(D) qPCR validation of TH levels in SN samples of PD and controls, normalized to beta-actin mRNA. T-test p*=0.025, Data presented as mean ± SD. n=18 for CT and 24 for PD.


Figure 2 ( D ) Heatmap of circRNA abundance , indicating significant tissue - based SN clustering , PD samples indicated in red .

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

Figure 2(D) Heatmap of circRNA abundance, indicating significant tissue-based SN clustering, PD samples indicated in red.


Figure 3 ( A ) Total circRNAs detected in the SN , MTG and AMG of both PD and control donors , p * = 0 . 02 p * = 0 . 026 and p * = 0 . 022 for comparison between PD and CT in SN , MTG and AMG . T - test for total SN vs MTG p * = 2 . 27E - 8 and SN vs AMG p * = 5 . 86E - 10 . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control and 13 for MTG PD , 10 for SN control and 15 for SN PD . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values . P value was calculated by T - test . ( B ) Global editing levels in the 3 brain regions is based on the Alu editing index , representing the weighted average editing level across all expressed Alu sequences , Wilcoxon , corrected p * = 0 . 038 , 0 . 055 and 0 . 42 for Amygdala , MTG and SN . Wilcoxon test between the Alu editing index of control and PD samples . ( C ) Global RNA editing levels based on the Alu editing index , studied only in Alu elements within circRNA exons and their flanking introns , in the 3 brain regions , for circRNAs corrected p * = 0 . 025 and 0 . 040 for the AMG and MTG and p = 0 . 3 for SN . For flanking introns p * = 0 . 02 and 0 . 040 for AMG and MTG and p = 0 . 34 for SN , Wilcoxon test . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control and 13 for MTG PD , 10 for SN control and 15 for SN PD . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values . ( D ) Negative correlation between total RNA editing and circRNA abundance , correlation = - 0 . 37 , Correlation test p value = 0 . 0015 . ( E ) Adar1 expression levels in all tissues . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values . ( F , G ) Correlations between circRNAs detected in each sample and the age of the control ( green , correlation = 0 . 68 , p - value = 0 . 032 ) and PD donors ( blue , correlation = - 0 . 14 p value = 0 . 62 ) . ( H ) Volcano plot of DE circRNAs in PD vs control tissues , red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test ( DEseq2 analysis ) . CircSLC8A1 is marked is red .

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

Figure 3(A) Total circRNAs detected in the SN, MTG and AMG of both PD and control donors, p*=0.02 p*=0.026 and p*=0.022 for comparison between PD and CT in SN, MTG and AMG. T-test for total SN vs MTG p*=2.27E-8 and SN vs AMG p*=5.86E-10. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values. P value was calculated by T-test.(B) Global editing levels in the 3 brain regions is based on the Alu editing index, representing the weighted average editing level across all expressed Alu sequences, Wilcoxon, corrected p*=0.038, 0.055 and 0.42 for Amygdala, MTG and SN. Wilcoxon test between the Alu editing index of control and PD samples.(C) Global RNA editing levels based on the Alu editing index, studied only in Alu elements within circRNA exons and their flanking introns, in the 3 brain regions, for circRNAs corrected p*= 0.025 and 0.040 for the AMG and MTG and p=0.3 for SN. For flanking introns p*= 0.02 and 0.040 for AMG and MTG and p=0.34 for SN, Wilcoxon test. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values.(D) Negative correlation between total RNA editing and circRNA abundance, correlation = -0.37, Correlation test p value = 0.0015.(E) Adar1 expression levels in all tissues. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values.(F,G) Correlations between circRNAs detected in each sample and the age of the control (green, correlation = 0.68, p-value = 0.032) and PD donors (blue, correlation = -0.14 p value = 0.62).(H) Volcano plot of DE circRNAs in PD vs control tissues, red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test (DEseq2 analysis). CircSLC8A1 is marked is red.


Figure 4 ( B , C ) CircSLC8A1 and linear SLC8A1 levels in the SN of control and PD brains , T - test p = 0 . 025 , p = 0 . 118 respectively . ( D ) qPCR validations of circSLC8A1 and SLC8A1 mRNA changes in the SN , T - test p * = 0 . 025 . n = 18 for CT and 24 for PD , x defines mean values . ( E ) no difference in circSLC8A1 levels in females and males , T - test p = 0 . 583 for control and p = 0 . 262 for PD . n = 15 females and 16 males . . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . ( F , G ) CircSLC8A1 and linear SLC8A1 mRNA levels are correlated in control , R2 = 0 . 47 T - test p = 0 . 0017 but not in PD samples , R2 = 0 . 016 , p > 0 . 05 . n = 18 for CT and 23 for PD . ( H ) Increased CircSLC8A1 RNA levels in PQ - exposed SH - SY cells ( normalized to Tubb3 and RPL19 ) while other circRNAs remain unchanged ( I , J ) Immunoblot decline ( western blot and quantification ) of the SLC8A1 protein in PQ - exposed SH - SY cells , with Tubulin as a loading control , T - test p * = 0 . 015 . n = 3 biological replicas for each condition . Data presented as mean ± SD .

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

Figure 4(B, C) CircSLC8A1 and linear SLC8A1 levels in the SN of control and PD brains, T-test p=0.025, p= 0.118 respectively.(D) qPCR validations of circSLC8A1 and SLC8A1 mRNA changes in the SN, T-test p*=0.025. n=18 for CT and 24 for PD, x defines mean values.(E) no difference in circSLC8A1 levels in females and males, T-test p=0.583 for control and p=0.262 for PD. n=15 females and 16 males. . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average.(F, G) CircSLC8A1 and linear SLC8A1 mRNA levels are correlated in control, R2=0.47 T-test p=0.0017 but not in PD samples, R2= 0.016, p>0.05. n=18 for CT and 23 for PD.(H) Increased CircSLC8A1 RNA levels in PQ-exposed SH-SY cells (normalized to Tubb3 and RPL19) while other circRNAs remain unchanged(I,J) Immunoblot decline (western blot and quantification) of the SLC8A1 protein in PQ-exposed SH-SY cells, with Tubulin as a loading control, T-test p*=0.015. n=3 biological replicas for each condition. Data presented as mean ± SD.


Figure 5 ( A ) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH - SY treatment of statins or the PF LRRK2 inhibitor , T - test p * * = 0 . 007 and p * * = 0 . 01 for PF - 06447475 and p * = 0 . 047 and p = * 0 . 0136 for Statins for cirSLC8A1 and SLC8A1 mRNA respectively ( B , C ) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures , T - test p * = 0 . 027 for Statins and p * = 0 . 011 for PF - 06447475 , n = 3 biological replicas for each condition . Data presented as mean ± SD . ( D ) SLC8A1 immunostaining of statin - treated SH - SY cells .

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

Figure 5(A) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH-SY treatment of statins or the PF LRRK2 inhibitor, T-test p**=0.007 and p**=0.01 for PF-06447475 and p*=0.047 and p=*0.0136 for Statins for cirSLC8A1 and SLC8A1 mRNA respectively(B,C) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures, T-test p*=0.027 for Statins and p*=0.011 for PF-06447475, n=3 biological replicas for each condition. Data presented as mean ± SD.(D) SLC8A1 immunostaining of statin-treated SH-SY cells.


Figure 6 ( B ) qPCR quantification from nuclear / cytoplasmic fractionations of SH - SY cells , T - test p = 0 . 005 for circSLC8A1 , p = 0 . 009 for NEAT1 and p = 0 . 009 for Rpl19 . n = 3 biological replicas for each condition . ( C ) qPCR quantification of Ago2 - bound RNA , immunoprecipitated ( IP ) / Input values normalized to negative control for a known Ago2 target , circCDR1as and circSLC8A1 , T - test p = 0 . 029 for circSLC8A1 and p = 0 . 001 for circCDR1as , n = 3 biological replicas for each condition . ( G ) relative expression of known miR - 128 targets from SN RNA - seq , p * * = 0 . 0004 for BMI1 , p * = 0 . 011 for SIRT1 and p * = 0 . 005 for AXIN1 . ( H ) relative expression of known miR - 128 targets in SH - SY cells treated with Paraquat , T - test p * = 0 . 03 , p * = 0 . 02 and p * = 0 . 002 for AXIN1 , BM1 and SIRT1 respectively , Data presented as mean ± SD with 4 - 6 biological replicas for each condition .

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 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 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

Figure 6(B) qPCR quantification from nuclear/cytoplasmic fractionations of SH-SY cells, T-test p=0.005 for circSLC8A1, p=0.009 for NEAT1 and p=0.009 for Rpl19. n=3 biological replicas for each condition.(C) qPCR quantification of Ago2-bound RNA, immunoprecipitated (IP)/Input values normalized to negative control for a known Ago2 target, circCDR1as and circSLC8A1, T-test p=0.029 for circSLC8A1 and p=0.001 for circCDR1as, n=3 biological replicas for each condition.(G) relative expression of known miR-128 targets from SN RNA-seq, p**=0.0004 for BMI1, p*=0.011 for SIRT1 and p*=0.005 for AXIN1.(H) relative expression of known miR-128 targets in SH-SY cells treated with Paraquat, T-test p*=0.03, p*=0.02 and p*=0.002 for AXIN1, BM1 and SIRT1 respectively, Data presented as mean ± SD with 4-6 biological replicas for each condition.


Figure 1A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population ( www . genenetwork . org ) . Adr : Adrenal Gland mRNA , Bla : Bladder mRNA , Bone : Bone Tibia mRNA , CNS : CNS mRNA , Cec : Cecum mRNA , Epi : Epididymis mRNA , Eso : Esophagus mRNA , Eye : Eye mRNA , Fat : Peritoneal Fat mRNA , GI : GI Track mRNA , Hea : Heart mRNA , Kid : Kidney mRNA , Liv : Liver mRNA , Lung : Lung mRNA , Mus : Gastrocnemius Muscle mRNA , Ova : Ovary mRNA , Pro : Prostate mRNA , Sal : Salivary Gland mRNA , Skin : Skin Back mRNA , Sn : Sciatic Nerve mRNA , Spl : Spleen mRNA , Tes : Testis mRNA , Thy : Thymus mRNA , Ton : Tongue mRNA , Tra : Trachea mRNA , and Ute : Uterus mRNA . B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population ( www . genenetwork . org ) . C Oocytes injected with cRNA encoding KCC4 were incubated with NAD + 200 μM and NAM 10 mM for 4 h . Lysates were immunoprecipitated with an anti - KCC4 antibody and then analyzed by SDS - PAGE / immunoblotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - injected oocytes ( UI ) were used as a control . D Oocytes expressing KCC4 were incubated with NAM for the indicated times , and KCC4 protein levels ( upper panel ) , and activity ( lower panel ) were analyzed by SDS - PAGE / immunoblotting or Cl - - dependent 86Rb + uptake under hypotonic conditions . E Oocytes injected with H20 or KCC4 were incubated for the indicated times , and KCC4 protein levels were analyzed by SDS - PAGE / immunoblotting . F Oocytes were injected with either WT , K114R or K114Q KCC4 and incubated with vehicle ( V ) , NAD + or NAM for 48 h , and KCC4 protein levels ( upper pannel ) , and activity ( lower pannel ) were analyzed as in D .

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 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 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 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 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

Figure 1A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population (www.genenetwork.org). Adr: Adrenal Gland mRNA, Bla: Bladder mRNA, Bone: Bone Tibia mRNA, CNS: CNS mRNA, Cec: Cecum mRNA, Epi: Epididymis mRNA, Eso: Esophagus mRNA, Eye: Eye mRNA, Fat: Peritoneal Fat mRNA, GI: GI Track mRNA, Hea: Heart mRNA, Kid: Kidney mRNA, Liv: Liver mRNA, Lung: Lung mRNA, Mus: Gastrocnemius Muscle mRNA, Ova: Ovary mRNA, Pro: Prostate mRNA, Sal: Salivary Gland mRNA, Skin: Skin Back mRNA, Sn: Sciatic Nerve mRNA, Spl: Spleen mRNA, Tes: Testis mRNA, Thy: Thymus mRNA, Ton: Tongue mRNA, Tra: Trachea mRNA, and Ute: Uterus mRNA.B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population (www.genenetwork.org).C Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS-PAGE/immunoblotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control.D Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS-PAGE/immunoblotting or Cl--dependent 86Rb+ uptake under hypotonic conditions.E Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS-PAGE/immunoblotting.F Oocytes were injected with either WT, K114R or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in D.


Figure 2A , B KCC4 activity ( A ) , and protein levels ( B ) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD + . Non - injected oocytes ( UI ) were used as a control . C KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild - type ( SIRT7 WT ) or the catalytically inactive SIRT7 ( SIRT7 H188Y ) for 48 h . D Oocytes were injected with either KCC4 WT , K114R or K114Q for 48 h and then incubated with vehicle ( V ) , NAD + or NAM for 24 h , and KCC4 and SIRT7 protein levels were analyzed by SDS - PAGE / immunoblotting . E Oocytes were injected with either KCC4 WT , K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle ( V ) or NAM for 4 h . After lysis , KCC4 was immunoprecipitated using an anti - KCC4 antibody , and the acetylation status of KCC4 was determined by SDS - PAGE / Western blotting using and anti - acetyl lysine antibody . F , G FLAG - KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD + ( F ) or NAM ( G ) and then analyzed by SDS - PAGE / immunoblotting using an anti - KCC4 or anti - SIRT7 antibody . Non - transfected cells ( UT ) were used as a control . H FLAG - KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD + and NAM . After lysis , KCC4 immunoprecipitation using an anti - FLAG antibody was performed , and the interaction with SIRT7 was determined by SDS - PAGE / Western blotting . I HEK293 cells expressing KCC4 were incubated with NAD + 200 μM and cycloheximide ( CHX ) for 0 , 3 , 6 and 9 h . KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA ( + SIRT7 ) or a shRNA against SIRT7 ( - SIRT7 ) .

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 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 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 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 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

Figure 2A, B KCC4 activity (A), and protein levels (B) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD+. Non-injected oocytes (UI) were used as a control.C KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild-type (SIRT7 WT) or the catalytically inactive SIRT7 (SIRT7 H188Y) for 48 h.D Oocytes were injected with either KCC4 WT, K114R or K114Q for 48 h and then incubated with vehicle (V), NAD+ or NAM for 24 h, and KCC4 and SIRT7 protein levels were analyzed by SDS-PAGE/immunoblotting.E Oocytes were injected with either KCC4 WT, K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle (V) or NAM for 4 h. After lysis, KCC4 was immunoprecipitated using an anti-KCC4 antibody, and the acetylation status of KCC4 was determined by SDS-PAGE/Western blotting using and anti-acetyl lysine antibody.F, G FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ (F) or NAM (G) and then analyzed by SDS-PAGE/immunoblotting using an anti-KCC4 or anti-SIRT7 antibody. Non-transfected cells (UT) were used as a control.H FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ and NAM. After lysis, KCC4 immunoprecipitation using an anti-FLAG antibody was performed, and the interaction with SIRT7 was determined by SDS-PAGE/Western blotting.I HEK293 cells expressing KCC4 were incubated with NAD+ 200 μM and cycloheximide (CHX) for 0, 3, 6 and 9 h. KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA (+SIRT7) or a shRNA against SIRT7 (-SIRT7).


Figure 3A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4 . After lysis , KCC4 was immunoprecipitated using an anti - FLAG antibody , and then analyzed by SDS - PAGE / Western blotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - transfected cells ( UT ) were used as a control . B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA ( - ) or a shRNA against SIRT7 ( + ) , and KCC4 activity and protein level were determined at the indicated pH . SIRT7 expression in these conditions is presented in C . C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells . At a lower pH , SIRT7 appears to translocate to the cytoplasm .

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 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

Figure 3A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4. After lysis, KCC4 was immunoprecipitated using an anti-FLAG antibody, and then analyzed by SDS-PAGE/Western blotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-transfected cells (UT) were used as a control.B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA (-) or a shRNA against SIRT7 (+), and KCC4 activity and protein level were determined at the indicated pH. SIRT7 expression in these conditions is presented in C.C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells. At a lower pH, SIRT7 appears to translocate to the cytoplasm.


Figure 4A , B Sirt7 mRNA ( A ) and protein ( B ) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau , respectively . Enrichment for each segment was previously validated by immunoblotting against segment - specific markers as indicated in material and methods ( Spirli et al , 2019 ) . C Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis ( SIRT7 , red ; ATPase , green ; DAPI , Blue ) . Scale bar : 20 μm . Controls of the specificity of antibodies using kidney of SIRT7 - deficient mice are provided in Appendix Fig S4 . D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis . Values are the mean ± SEM .

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 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 4A, B Sirt7 mRNA (A) and protein (B) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau, respectively. Enrichment for each segment was previously validated by immunoblotting against segment-specific markers as indicated in material and methods (Spirli et al, 2019).C Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis (SIRT7, red; ATPase, green; DAPI, Blue). Scale bar: 20 μm. Controls of the specificity of antibodies using kidney of SIRT7-deficient mice are provided in Appendix Fig S4.D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis. Values are the mean ± SEM.


Figure 5A KCC4 protein levels were determined in the kidneys of wild - type ( SIRT7 + / + ) and SIRT7 - deficient mice ( SIRT7 - / - ) . SIRT7 protein expression are absent in the kidneys of SIRT7 - deficient mice . B KCC4 protein levels , acetylation status of KCC4 , and interaction with SIRT7 in kidneys of SIRT7 + / + and SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis . C Immunolocalization of KCC4 and H - ATPase in the kidneys of SIRT7 + / + and SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis ( NH4Cl ) . ( KCC4 , red ; H - ATPase , green ) . Scale bar : 20 μm . Cell count was performed automatically using the Gen5 software to report the percentage of KCC4 + cells among H - ATPase + cells ( lower panel ) . D NCC and NKCC2 protein levels in the kidney of SIRT7 + / + and SIRT7 - / - mice under basal conditions .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A KCC4 protein levels were determined in the kidneys of wild-type (SIRT7+/+) and SIRT7-deficient mice (SIRT7-/-). SIRT7 protein expression are absent in the kidneys of SIRT7-deficient mice.B KCC4 protein levels, acetylation status of KCC4, and interaction with SIRT7 in kidneys of SIRT7+/+ and SIRT7-/-mice under normal conditions (Control) or with metabolic acidosis.C Immunolocalization of KCC4 and H-ATPase in the kidneys of SIRT7+/+ and SIRT7-/- mice under normal conditions (Control) or with metabolic acidosis (NH4Cl). (KCC4, red; H-ATPase, green). Scale bar: 20 μm. Cell count was performed automatically using the Gen5 software to report the percentage of KCC4+ cells among H-ATPase+ cells (lower panel).D NCC and NKCC2 protein levels in the kidney of SIRT7+/+ and SIRT7-/- mice under basal conditions.


Figure 1HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( A ) Concentrations of IL - 6 were determined ( CTRL , n = 9 ; Bax / Bak , n = 6 ; Bcl - XL , n = 3 ; STING , n = 3 ) . HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( B ) Concentrations of IL - 8 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) . HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( C ) Concentrations of CXCL1 were determined ( CTRL , n = 8 ; Bax / Bak , n = 5 ) . HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( D ) Concentrations of FGF - 2 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) . HeLa cell lines were treated with the Mcl - 1 - inhibitor S63845 for 72 h . Cytokines were measured by ELISA . ( E ) HeLa CTRL cells and Bax / Bak - deficient cells were treated with the Mcl - 1 - inhibitor S63845 . Concentrations of IL - 6 in supernatants are shown ( CTRL , n = 8 ; Bax / Bak , n = 7 ) . F 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing Bcl - XL were treated with ABT - 737 . Concentrations of IL - 6 in the supernatants were measured after 48 h ( n = 5 ) . G 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing mouse Bcl - XL were treated with various concentrations of ABT - 737 . To some aliquots the caspase - inhibitor zVAD - fmk ( 50 μM ) was added . Relative numbers of apoptotic cells were determined as in EV 1B after 48 h . Data are means / SEM of at least three independent experiments . ( 1205Lu , n = 4 ; 1205Lu 5 µM ABT - 737 , n = 3 ; 1205Lu / Bcl - XL and 1205Lu + zVAD , n = 3 ) . H HeLa - UL12 . 5 cells untreated or treated with tamoxifen ( 100 nM , 24 h ) were stained with Picogreen ( 3 µl / ml ) to detect mitochondrial DNA . Scale bare , 10 µmI HeLa CTRL cells and HeLa - UL12 . 5 cells were treated with ABT - 737 ( 10µM ) . Concentration of IL - 6 in the supernatant were measured after 72h ( n = 3 ) .

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

Figure 1HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (A) Concentrations of IL-6 were determined (CTRL, n=9; Bax/Bak, n=6; Bcl-XL, n=3; STING, n=3).HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (B) Concentrations of IL-8 were determined (CTRL, n=7; Bax/Bak, n=4).HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (C) Concentrations of CXCL1 were determined (CTRL, n=8; Bax/Bak, n=5).HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (D) Concentrations of FGF-2 were determined (CTRL, n=7; Bax/Bak, n=4).HeLa cell lines were treated with the Mcl-1-inhibitor S63845 for 72 h. Cytokines were measured by ELISA. (E) HeLa CTRL cells and Bax/Bak-deficient cells were treated with the Mcl-1-inhibitor S63845. Concentrations of IL-6 in supernatants are shown (CTRL, n=8; Bax/Bak, n=7).F 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing Bcl-XL were treated with ABT-737. Concentrations of IL-6 in the supernatants were measured after 48 h (n=5).G 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing mouse Bcl-XL were treated with various concentrations of ABT-737. To some aliquots the caspase-inhibitor zVAD-fmk (50 μM) was added. Relative numbers of apoptotic cells were determined as in EV 1B after 48 h. Data are means/SEM of at least three independent experiments. (1205Lu, n=4; 1205Lu 5 µM ABT-737, n=3; 1205Lu/Bcl-XL and 1205Lu + zVAD, n=3).H HeLa-UL12.5 cells untreated or treated with tamoxifen (100 nM, 24 h) were stained with Picogreen (3 µl/ml) to detect mitochondrial DNA. Scale bare, 10 µmI HeLa CTRL cells and HeLa-UL12.5 cells were treated with ABT-737 (10µM). Concentration of IL-6 in the supernatant were measured after 72h (n=3).


Figure 2A , The caspase - 3 - reporter cell line ( labelled DEVD ) and the control cell line ( DEVG ; see methods ) were infected and analysed at various time points post - infection by flow cytometry ( Chlamydia trachomatis ( Ctr ; MOI = 1 , 30 h ) ; Salmonella Typhimurium ( STy ; MOI = 50 , 24h ) ; Toxoplasma gondii ( MOI = 5 , 16 h ) ; MVA ( MOI = 10 , 16h ) ; Influenza A virus ( IAV ; MOI = 1 , 16h ) ; Herpes simplex virus 1 ( HSV - 1 ; MOI = 1 , 16h ) . Staurosporine treated cells ( 1 μM , 3h ) were used as a positive control . Percentages of cells positive for caspase - 3 - activation ( FRET - loss ) were determined . Data are means / SEM of three independent experiments . ( n = 3 ) B , C Cells were infected with modified vaccinia virus Ankara ( MVA ) ( MOI = 10 ) . The caspase - inhibitor zVAD - fmk was used at 100µM ; cells were analysed 16 h post - infection . D , E Cells were infected with influenza A virus ( IAV ) ( MOI = 1 ; analysis was 16 h post - infection ) . D , detection of viral M2 protein was used as infection control . F , G Cells were infected with Herpes simplex virus 1 ( HSV - 1 ) ( MOI = 1 ; analysis was 16 h post - infection ) . H Cells were infected with Chlamydia trachomatis ( Ctr ) ( MOI = 1 ; analysis was 30 h post - infection ) . I Cells were infected with Salmonella Typhimurium ( Sty ) ( MOI = 50 ; analysis was 24 h post - infection ) . J Cells were infected with the protozoan parasite Toxoplasma gondii ( MOI = 5 ; analysis was 16 h post - infection ) .

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 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 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 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 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

Figure 2A, The caspase-3-reporter cell line (labelled DEVD) and the control cell line (DEVG; see methods) were infected and analysed at various time points post-infection by flow cytometry (Chlamydia trachomatis (Ctr; MOI=1, 30 h); Salmonella Typhimurium (STy; MOI=50, 24h); Toxoplasma gondii (MOI=5, 16 h); MVA (MOI=10, 16h); Influenza A virus (IAV; MOI=1, 16h); Herpes simplex virus 1 (HSV-1; MOI=1, 16h). Staurosporine treated cells (1 μM, 3h) were used as a positive control. Percentages of cells positive for caspase-3-activation (FRET-loss) were determined. Data are means/SEM of three independent experiments. (n=3)B, C Cells were infected with modified vaccinia virus Ankara (MVA) (MOI=10). The caspase-inhibitor zVAD-fmk was used at 100µM; cells were analysed 16 h post-infection.D, E Cells were infected with influenza A virus (IAV) (MOI=1; analysis was 16 h post-infection). D, detection of viral M2 protein was used as infection control.F, G Cells were infected with Herpes simplex virus 1 (HSV-1) (MOI=1; analysis was 16 h post-infection).H Cells were infected with Chlamydia trachomatis (Ctr) (MOI=1; analysis was 30 h post-infection).I Cells were infected with Salmonella Typhimurium (Sty) (MOI=50; analysis was 24 h post-infection).J Cells were infected with the protozoan parasite Toxoplasma gondii (MOI=5; analysis was 16 h post-infection).


Figure 3A Infection with MVA ( MOI = 10 for 16 h ; IL - 6 ) . B infection with HSV - 1 ( MOI = 1 for 16 h ; IL - 6 ) . Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; IL - 6Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; , IL - 8 ) . Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; IL - 6Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; CXCL - 1 ) . Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; IL - 6Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; , IL - 8Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; CXCL - 1Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; FGF - 2 ) .

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

Figure 3A Infection with MVA (MOI=10 for 16 h; IL-6).B infection with HSV-1 (MOI=1 for 16 h; IL-6).Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; IL-6Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; , IL-8).Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; IL-6Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; CXCL-1).Infection with Toxoplasma gondii (MOI=5 for 16 h; IL-6Infection with Toxoplasma gondii (MOI=5 for 16 h; , IL-8Infection with Toxoplasma gondii (MOI=5 for 16 h; CXCL-1Infection with Toxoplasma gondii (MOI=5 for 16 h; FGF-2).


Figure 4 ( A ) HeLa cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of six experiments . ( A ) Growth of C . trachomatis was measured in HeLa cells as increase in GFP - fluorescence between 18 and 38 h . The data show means / SEM of six independent experiments ( genotype effect ( significance of the difference of GFP - expression between the cell lines overall , p = 0 . 08 ) . Red line indicates the fold change calculation of the individual different time points and cell lines ( significance tested ( one sample t - test ) for each time point for a difference between Bax / Bak vs CTRL ) . AGS cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of 4 - 5 experiments . ( B ) Growth of C . trachomatis was measured in AGS cells as increase in GFP - fluorescence . The data show means / SEM of 4 to 5 independent experiments ( genotype effect : p < 0 . 0001 ; time - genotype interaction : p < 0 . 0001 ) ( 12 h and 38 h , n = 4 ; 18 h , n = 5 ) . C Chlamydial genomes per cell culture ( expressed as chlamydial DNA in 50 ng of total DNA , determined by quantitative PCR ) . * , p < 0 . 05 between control and mutant cells . ( n = 3 ) D CTRL , Bax / Bak - deficient or Bcl - XL - over - expressing HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . Extracellular bacteria were killed using non cell - permeable antibiotic . At the indicated time points cells were lysed , and aliquots were plated on agar plates . Recovered colony forming units were calculated . Data are means / SEM of four independent experiments . Significant genotype effects were found between mutant and control cells ( Bax / Bak p < 0 . 0001 ; Bcl - XL p 0 . 0001 ) . E CTRL , Bax / Bak - deficient or Apaf - 1 - deficient HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . CFU / well were calculated as in ( D ) . n = 3 .

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 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 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 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 4(A) HeLa cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of six experiments. (A) Growth of C. trachomatis was measured in HeLa cells as increase in GFP-fluorescence between 18 and 38 h. The data show means/SEM of six independent experiments (genotype effect (significance of the difference of GFP-expression between the cell lines overall, p = 0.08). Red line indicates the fold change calculation of the individual different time points and cell lines (significance tested (one sample t-test) for each time point for a difference between Bax/Bak vs CTRL).AGS cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of 4-5 experiments. (B) Growth of C. trachomatis was measured in AGS cells as increase in GFP-fluorescence. The data show means/SEM of 4 to 5 independent experiments (genotype effect: p<0.0001; time-genotype interaction: p<0.0001) (12 h and 38 h, n=4; 18 h, n=5).C Chlamydial genomes per cell culture (expressed as chlamydial DNA in 50 ng of total DNA, determined by quantitative PCR). *, p<0.05 between control and mutant cells. (n=3)D CTRL, Bax/Bak-deficient or Bcl-XL-over-expressing HeLa cells were infected with Salmonella Typhimurium (MOI=50). Extracellular bacteria were killed using non cell-permeable antibiotic. At the indicated time points cells were lysed, and aliquots were plated on agar plates. Recovered colony forming units were calculated. Data are means/SEM of four independent experiments. Significant genotype effects were found between mutant and control cells (Bax/Bak p<0.0001; Bcl-XL p 0.0001).E CTRL, Bax/Bak-deficient or Apaf-1-deficient HeLa cells were infected with Salmonella Typhimurium (MOI=50). CFU/well were calculated as in (D). n=3.


Figure 2A Actin expression levels shown by western blotting for strains expressing S . cerevisiae ' s actin protein from various coding sequences , with tubulin ( Tub1p ) as a loading control . B Quantification of actin expression levels , showing a decrease when more silent mutations are present . Data are presented as mean + / - SD ( n = 4 for Sc , n = 8 for ScNI ; n = 12 for Sc [ Ca ] , Sc [ Sp ] and Sc [ At ] ; 2 biological replicates with n / 2 technical replicates each ) . * P < 0 . 05 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . F In vivo actin network deviation indexes , defined to evaluate the patch - cable balance compared to S . cerevisiae haploid cells ( value is 0 in S . cerevisiae ' s cells , 1 when cells contain only actin patches and - 1 when cells contain only cables ) . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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

Figure 2A Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin protein from various coding sequences, with tubulin (Tub1p) as a loading control. B Quantification of actin expression levels, showing a decrease when more silent mutations are present. Data are presented as mean +/- SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).F In vivo actin network deviation indexes, defined to evaluate the patch-cable balance compared to S. cerevisiae haploid cells (value is 0 in S. cerevisiae's cells, 1 when cells contain only actin patches and -1 when cells contain only cables). Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


Figure 3E Phalloidin staining of F - actin organization . Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed . Micrographs of Sc and Sc _ NI cells are reproduced from Fig 2E . Scale bar : 3 µm . F In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . I Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm J Quantification of actin patch resistance to CK - 666 treatment . Bar graphs represent the percentage of cells with a given number of visible actin patches after CK - 666 treatment . ( n = 41 for ScNI , 55 for N2 , 31 for Ca , 41 for Op , 55 for Hs ) .

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 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

Figure 3E Phalloidin staining of F-actin organization. Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed. Micrographs of Sc and Sc_NI cells are reproduced from Fig 2E. Scale bar: 3 µm.F In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).I Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm J Quantification of actin patch resistance to CK-666 treatment. Bar graphs represent the percentage of cells with a given number of visible actin patches after CK-666 treatment. (n = 41 for ScNI, 55 for N2, 31 for Ca, 41 for Op, 55 for Hs).


Figure 4A ( Top ) Snapshots of actin networks assembled from three different actins sources : Act _ Sc , Act _ N2 and Act _ Ca . ( Bottom left ) Quantification of actin fluorescence on beads . Data are presented as mean + / - SD ( n = 52 for Act _ Sc branched , n = 70 for Act _ N2 branched , n = 81 for Act _ Ca branched , n = 32 for Act _ Sc linear , n = 34 for Act _ N2 linear and n = 47 for Act _ Ca linear ) . * * P < 0 . 01 , * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . ( Bottom right ) In vitro actin network deviation indexes . B ( Left ) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa - 488 - labeled ADF / cofilin ( replacement of unlabeled ADF / cofilin ) . ( Right ) Quantification of ADF / cofilin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 34 for Act _ Sc branched , n = 28 for Act _ N2 branched , n = 43 for Act _ Ca branched , n = 24 for Act _ Sc linear , n = 15 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . C ( Left ) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488 - tropomyosin . ( Right ) Quantification of tropomyosin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 45 for Act _ Sc branched , n = 63 for Act _ N2 branched , n = 57 for Act _ Ca branched , n = 22 for Act _ Sc linear , n = 32 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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 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 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

Figure 4A (Top) Snapshots of actin networks assembled from three different actins sources: Act_Sc, Act_N2 and Act_Ca. (Bottom left) Quantification of actin fluorescence on beads. Data are presented as mean +/- SD (n = 52 for Act_Sc branched, n = 70 for Act_N2 branched, n = 81 for Act_Ca branched, n = 32 for Act_Sc linear, n = 34 for Act_N2 linear and n = 47 for Act_Ca linear). **P<0.01, ***P<0.001 (Kruskal-Wallis test, with multiple comparisons). (Bottom right) In vitro actin network deviation indexes.B (Left) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa-488-labeled ADF/cofilin (replacement of unlabeled ADF/cofilin). (Right) Quantification of ADF/cofilin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 34 for Act_Sc branched, n = 28 for Act_N2 branched, n = 43 for Act_Ca branched, n = 24 for Act_Sc linear, n = 15 for Act_N2 linear and n = 27 for Act_Ca linear). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).C (Left) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488-tropomyosin. (Right) Quantification of tropomyosin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 45 for Act_Sc branched, n = 63 for Act_N2 branched, n = 57 for Act_Ca branched, n = 22 for Act_Sc linear, n = 32 for Act_N2 linear and n = 27 for Act_Ca linear). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


Figure 6C Phalloidin staining depicting F - actin organization . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . D In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . E Polarity Indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . F Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . G In vivo actin network deviation indexes of cells treated with DMSO or CK - 666 . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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 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

Figure 6C Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. D In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). E Polarity Indexes. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).F Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. G In vivo actin network deviation indexes of cells treated with DMSO or CK-666. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


Figure 1 ( C ) The 3 . 1 Å 2Fo - Fc map ( 1 . 2 σ ) around the DNA of the final refined ATPγS - MjMR - DNA complex structure . ( D ) A ribbon representation of the 3 . 1 Å ATPγS - MjMR - DNA structure showing the Mre11 nuclease , capping , and C - terminal three helix - bundle domain ( light pink ) , Rad50 molecules ( green and light blue ) , and the DNA ( yellow and orange ) . ATPγS is shown in spheres . See Movie EV 1 for the movement of the complex .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(C) The 3.1 Å 2Fo-Fc map (1.2 σ) around the DNA of the final refined ATPγS-MjMR-DNA complex structure.(D) A ribbon representation of the 3.1 Å ATPγS-MjMR-DNA structure showing the Mre11 nuclease, capping, and C-terminal three helix-bundle domain (light pink), Rad50 molecules (green and light blue), and the DNA (yellow and orange). ATPγS is shown in spheres. See Movie EV 1 for the movement of the complex.


Figure 2 ( A ) A ribbon diagram showing the overall DNA recognition by the ATPγS - Rad50 dimer . ( B ) Close - up view of the interaction between the internal segment ( major and minor grooves ) of the DNA strand and the edge of lobe I of Rad50A ( green ) . Loop α1 - α2 wedge faces the minor groove between the 10th and 13 ' th phosphates of the template ( orange ) and non - template ( yellow ) strands , respectively . ( C ) Close - up view of the interaction between the DNA strand and the α1 - α2 loop in an orientation different from Fig 2B .

O O B-PANEL_START 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

Figure 2(A) A ribbon diagram showing the overall DNA recognition by the ATPγS-Rad50 dimer.(B) Close-up view of the interaction between the internal segment (major and minor grooves) of the DNA strand and the edge of lobe I of Rad50A (green). Loop α1-α2 wedge faces the minor groove between the 10th and 13' th phosphates of the template (orange) and non-template (yellow) strands, respectively. (C) Close-up view of the interaction between the DNA strand and the α1-α2 loop in an orientation different from Fig 2B.


Figure 3 ( B , C ) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA ( φx174RFII ) in the absence ( B ) or presence ( C ) of AMP - PNP / Mg2 + . Each protein sample was incubated with φx174RFII dsDNA ( 3 . 5 nM ) for 30 min on 4 ° C . The following molar ratio of protein : DNA is used in Fig 3B - 3E ; lane 2 , 50 : 1 ; lane 3 , 100 : 1 ; lane 4 , 200 : 1 ; lane 5 , 500 : 1 ; lane 6 , 1000 : 1 ; lane 7 , 2000 : 1 . We note that some MRcd variants may form a second form of the complex with DNA . ( D , E ) Interactions of the WT or five full - length TmMR mutants with φx174RFII were examined in the absence ( D ) or presence of AMP - PNP / Mg2 + ( E ) . Reaction conditions are same as those in Fig 3B . ( F ) Nuclease activities of free MjMre11 ( lane 3 , 4 ) , nucleotide unbound - ( 5 , 6 ) and - bound MjMRcd complex ( 7 , 8 ) towards a hairpin DNA . Each protein sample was incubated with a hairpin DNA ( 10 nM ) in a 1 : 10 or 1 : 20 molar ratio ( protein : DNA ) for 30 min on 55 ° C . The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment . ( G ) Nuclease activities of free TmMre11 alone ( lane 3 , 4 ) , nucleotide unbound - ( lane 5 , 6 ) and - bound ( lane 7 , 8 ) full - length TmMR complex towards a hairpin DNA .

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 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 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 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

Figure 3(B, C) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA (φx174RFII) in the absence (B) or presence (C) of AMP-PNP/Mg2+. Each protein sample was incubated with φx174RFII dsDNA (3.5 nM) for 30 min on 4°C. The following molar ratio of protein: DNA is used in Fig 3B-3E; lane 2, 50:1; lane 3, 100:1; lane 4, 200:1; lane 5, 500:1; lane 6, 1000:1; lane 7, 2000:1. We note that some MRcd variants may form a second form of the complex with DNA.(D, E) Interactions of the WT or five full-length TmMR mutants with φx174RFII were examined in the absence (D) or presence of AMP-PNP/Mg2+ (E). Reaction conditions are same as those in Fig 3B.(F) Nuclease activities of free MjMre11 (lane 3, 4), nucleotide unbound- (5, 6) and -bound MjMRcd complex (7, 8) towards a hairpin DNA. Each protein sample was incubated with a hairpin DNA (10 nM) in a 1:10 or 1:20 molar ratio (protein: DNA) for 30 min on 55°C. The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment.(G) Nuclease activities of free TmMre11 alone (lane 3, 4), nucleotide unbound- (lane 5, 6) and -bound (lane 7, 8) full-length TmMR complex towards a hairpin DNA.


Figure 4 ( A ) Superposition of the ATPγS - MjMR - DNA complex ( green ) and apo - MjMR ( 3AV0 , magenta ) structures . Alignment was done using their NBDs ( 1 . 3 Å rmsd in the positions of 311 Cα atoms ) . The coiled - coils of the DNA - bound Rad50 molecules are shifted ( arrow ) to more parallel orientation . The entire structures can be superimposed with a 1 . 9 Å rmsd in 692 Cα positions . ( B , C ) Comparison of the local structures between the apo ( B ) and the DNA - bound MjMR complex ( C ) . Upon DNA binding , helix α6 translates to DNA backbone , resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II . Arrows indicate the direction of the helix movement by DNA binding . See Fig EV5 for a close - up view .

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 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(A) Superposition of the ATPγS-MjMR-DNA complex (green) and apo-MjMR (3AV0, magenta) structures. Alignment was done using their NBDs (1.3 Å rmsd in the positions of 311 Cα atoms). The coiled-coils of the DNA-bound Rad50 molecules are shifted (arrow) to more parallel orientation. The entire structures can be superimposed with a 1.9 Å rmsd in 692 Cα positions.(B, C) Comparison of the local structures between the apo (B) and the DNA-bound MjMR complex (C). Upon DNA binding, helix α6 translates to DNA backbone, resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II. Arrows indicate the direction of the helix movement by DNA binding. See Fig EV5 for a close-up view.


Figure 5 ( A ) Stereo view of the superposition of the ADP - MjRad50 ( 3AUX ) complex ( black ) onto the ATPγS - MjMR - DNA complex ( green and blue ) by aligning their lobe II domains . The α1 - α2 loop that could collide with parts of the DNA molecule is indicated . ( B ) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11 , which could allow the access of the unwound DNA . Color schemes are same as those in Fig 5A . ( C ) DNA - unwinding activities of TmMR . Lane 2 , no protein ; lanes 3 and 4 , no ATP ; lanes 5 and 6 , with ATPγS ; lanes 7 and 8 , with ATP . The DNA : protein ratio was 1 : 2 for lanes 3 , 5 , and 7 , and 1 : 5 for lanes 4 , 6 , and 8 . Plotted data represent the mean of three experiments , with error bars indicating one standard deviation . A graph of the quantified data is shown in the lower panel . ( D ) Comparison of the ATP - dependent DNA unwinding activities of WT and mutant TmMR proteins . Lane 2 , no protein ; lane 3 and 4 , WT TmMR ; lane 5 and 6 , Rad50 ( Δ54 - 56 ) mutant ; lane 7 and 8 , R87E mutant ; lane 9 and 10 , K95E mutant ; lane 11 and 12 , R94E / K95E mutant ; lane 13 and 14 , K115E mutant . For each TmMR , reaction contains 1 : 2 or 1 : 5 ratio of DNA : protein .

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

Figure 5(A) Stereo view of the superposition of the ADP-MjRad50 (3AUX) complex (black) onto the ATPγS-MjMR-DNA complex (green and blue) by aligning their lobe II domains. The α1-α2 loop that could collide with parts of the DNA molecule is indicated. (B) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11, which could allow the access of the unwound DNA. Color schemes are same as those in Fig 5A.(C) DNA-unwinding activities of TmMR. Lane 2, no protein; lanes 3 and 4, no ATP; lanes 5 and 6, with ATPγS; lanes 7 and 8, with ATP. The DNA: protein ratio was 1:2 for lanes 3, 5, and 7, and 1:5 for lanes 4, 6, and 8. Plotted data represent the mean of three experiments, with error bars indicating one standard deviation. A graph of the quantified data is shown in the lower panel.(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.


Figure 6 ( A ) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence ( left panel ) or presence of ATP ( right panel ) . Major products are marked with dots . The 5 ' - 32P - label is indicated by a star . See Appendix Fig S4A and S4B for the quantified values . ( B ) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP - PNP . See Appendix Fig S3E and S3F for the original data . ( C ) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence ( left panel ) or presence of ATP ( right panel ) . See Appendix Fig S4C and S4D for the quantified values . ( D ) The effects of single or multiple point mutations in the DNA - contacting or neighboring residues of S . cerevisiae rad50 on the sensitivity to chemical drugs . Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT , PHL , and MMS were examined . ( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values . ( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values .

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 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 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

Figure 6(A) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence (left panel) or presence of ATP (right panel). Major products are marked with dots. The 5'-32P-label is indicated by a star. See Appendix Fig S4A and S4B for the quantified values.(B) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP-PNP. See Appendix Fig S3E and S3F for the original data.(C) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence (left panel) or presence of ATP (right panel). See Appendix Fig S4C and S4D for the quantified values.(D) The effects of single or multiple point mutations in the DNA-contacting or neighboring residues of S. cerevisiae rad50 on the sensitivity to chemical drugs. Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT, PHL, and MMS were examined. (E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.(E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


Figure 1B ) Single point activity assays of USP1WT and deletion mutants on Ub - Rho show signifi cantly increased activity in mutants where insert L1 and L3 are both deleted . C ) Single point activity assays of USP1WT and deletion mutants ( ± UAF1 ) on Ub - Rho show the loss in hyper - activation of USP1ΔL1L3 upon addition of UAF1 . D ) Michaelis - Menten analysis of USP1 deletion mutants against ubiquitin - rhodamine ( Ub - Rho ) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants ( n = 2 , biological replicates ) . E ) Michaelis - Menten analysis of USP1 deletion mutants ( + UAF1 ) against Ub - Rho shows that all deletions mutants have similar catalytic activity ( n = 2 , biological replicates ) . F ) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i . e . LRGG - AMC ( 100µM ) . USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT ( n = 2 , technical replicates ) . G ) Comparing activity of USP1WT and USP1ΔL1L3 ( + UAF1 ) on a peptide substrate i . e . LRGG - AMC ( 100µM ) . Addition of UAF1 allows cleavage of LRGG - AMC by both USP1WT and USP1ΔL1L3 ( n = 2 , technical replicates ) .

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

Figure 1B) Single point activity assays of USP1WT and deletion mutants on Ub-Rho show significantly increased activity in mutants where insert L1 and L3 are both deleted.C) Single point activity assays of USP1WT and deletion mutants (± UAF1) on Ub-Rho show the loss in hyper-activation of USP1ΔL1L3 upon addition of UAF1.D) Michaelis-Menten analysis of USP1 deletion mutants against ubiquitin-rhodamine (Ub-Rho) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants (n=2, biological replicates).E) Michaelis-Menten analysis of USP1 deletion mutants (+UAF1) against Ub-Rho shows that all deletions mutants have similar catalytic activity (n=2, biological replicates).F) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i.e. LRGG-AMC (100µM). USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT (n=2, technical replicates).G) Comparing activity of USP1WT and USP1ΔL1L3 (+UAF1) on a peptide substrate i.e. LRGG-AMC (100µM). Addition of UAF1 allows cleavage of LRGG-AMC by both USP1WT and USP1ΔL1L3 (n=2, technical replicates).


Figure 2A ) Quantification of gel - based activity assays where the activity of USP1WT and deletion mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . The USP1ΔL1 mutant has reduced activity on PCNA - UbTAMRA compared to the other USP1 variants tested ( n = 2 , biological replicates ) . C ) Quantification of gel - based activity assays where the activity of USP1 PIP mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . Both the PIP mutants have reduced activity on PCNA - Ub compared to USP1WT ( n = 2 , biological replicates ) . D ) FP based binding assays of USP1 mutants ( + UAF1 ) with PCNA - UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT ( n = 2 , technical replicates ) . E ) Quantification of gel - based activity assays where the activity of USP1WT + UAF1 on PCNA - UbWT is compared with PCNA - UbPIM1 shows that PCNA - UbWT is cleaved much faster compared to PCNA - UbPIM1 ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) F ) Comparing activity of USP1WT and USP1PIP1 ( + UAF1 ) on PCNA - UbWT and PCNA - UbPIM1 in gel - based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA , whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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

Figure 2A) Quantification of gel-based activity assays where the activity of USP1WT and deletion mutants (+UAF1) on PCNA-UbTAMRA is compared. The USP1ΔL1 mutant has reduced activity on PCNA-UbTAMRA compared to the other USP1 variants tested (n=2, biological replicates).C) Quantification of gel-based activity assays where the activity of USP1 PIP mutants (+UAF1) on PCNA-UbTAMRA is compared. Both the PIP mutants have reduced activity on PCNA-Ub compared to USP1WT (n=2, biological replicates).D) FP based binding assays of USP1 mutants (+ UAF1) with PCNA-UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT (n=2, technical replicates).E) Quantification of gel-based activity assays where the activity of USP1WT+UAF1 on PCNA-UbWT is compared with PCNA-UbPIM1 shows that PCNA-UbWT is cleaved much faster compared to PCNA-UbPIM1 (n=2, technical replicates (example shown here from 1 of 2 biological replicates))F) Comparing activity of USP1WT and USP1PIP1 (+UAF1) on PCNA-UbWT and PCNA-UbPIM1 in gel-based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA, whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).


Figure 3A ) Schematic representation of loading and purification of PCNA - Ub on nicked circular DNA . The DNA - loaded PCNA - Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA - loaded PCNA - Ub . B ) Michaelis - Menten analysis of USP1 + UAF1 with and without DNA ( 65bp dsDNA ) shows that DNA binding alone has no effect on USP1 activity ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) . C ) Coomassie stained gel of in vitro activity assay showing increased activity of USP1 - UAF1 on DNA - loaded PCNA - Ub compared to PCNA - Ub ( - DNA ) and PCNA - Ub ( + DNA , + RFC and not ATP ) . D ) Quantification of gel - based activity assays showing enhanced activity of USP1 + UAF1 on DNA - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) . E ) Quantification of gel - based activity assays showing enhanced activity of USP1 on DNA - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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 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 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 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 3A) Schematic representation of loading and purification of PCNA-Ub on nicked circular DNA. The DNA-loaded PCNA-Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA-loaded PCNA-Ub.B) Michaelis-Menten analysis of USP1+UAF1 with and without DNA (65bp dsDNA) shows that DNA binding alone has no effect on USP1 activity (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).C) Coomassie stained gel of in vitro activity assay showing increased activity of USP1-UAF1 on DNA-loaded PCNA-Ub compared to PCNA-Ub (-DNA) and PCNA-Ub (+DNA, +RFC and not ATP).D) Quantification of gel-based activity assays showing enhanced activity of USP1+UAF1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).E) Quantification of gel-based activity assays showing enhanced activity of USP1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).


Figure 4A ) Gel based quantification of USP1 - UAF1 activity on PCNA - Ub ( DNA loaded and free ) for three different concentration of enzyme and substrate . B ) FP based activity assays on free PCNA - Ub ( three concentrations of USP1 and PCNA - Ub ) and activity of USP1 on increasing concentrations of Ub - Rho .

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

Figure 4A) Gel based quantification of USP1-UAF1 activity on PCNA-Ub (DNA loaded and free) for three different concentration of enzyme and substrate.B) FP based activity assays on free PCNA-Ub (three concentrations of USP1 and PCNA-Ub) and activity of USP1 on increasing concentrations of Ub-Rho.


Figure 5A ) Comparison of USP1WT and deletion mutants ( + UAF1 ) for activity on DNA - loaded PCNA - Ub ( solid lines ) and free PCNA - Ub ( dashed lines ) shows no increase in USP1ΔL1 activity on the loaded substrate . Left panel : Quantification of gel - based activity assays showing percentage of cleaved PCNA - Ub at the mentioned time points ( n = 2 , biological replicates ) , Right panel : Quantification of the activation fold observed in USP1WT and mutants on DNA - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , biological replicates ) . B ) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species . Three groups of positively charged residues ( * * * ) are mutated separately and tested for binding to double stranded DNA ( 65bp ) using SPR which shows the importance of this region for DNA binding of USP1 . C ) Comparison of USP1DM1 , USP1PIP1 and USP1PIP1 + DM1 ( + UAF1 ) activity on DNA - loaded PCNA - Ub ( solid lines ) and free PCNA - Ub ( dashed lines ) shows reduced increase in activity of these mutants compared to USP1WT . Left panel : Quantification of gel based activity assays showing percentage of cleaved PCNA - Ub at the mentioned time points ( n = 2 , technical replicates ) , Right panel : Quantification of the activation fold observed in USP1WT , USP1PIP1 , USP1DM1 and USP1PIP1 + DM1 on DNA - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , technical replicates ) . D ) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA .

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

Figure 5A) Comparison of USP1WT and deletion mutants (+UAF1) for activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows no increase in USP1ΔL1 activity on the loaded substrate. Left panel: Quantification of gel-based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, biological replicates), Right panel: Quantification of the activation fold observed in USP1WT and mutants on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, biological replicates).B) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species. Three groups of positively charged residues (***) are mutated separately and tested for binding to double stranded DNA (65bp) using SPR which shows the importance of this region for DNA binding of USP1.C) Comparison of USP1DM1, USP1PIP1 and USP1PIP1+DM1 (+UAF1) activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows reduced increase in activity of these mutants compared to USP1WT. Left panel: Quantification of gel based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, technical replicates), Right panel: Quantification of the activation fold observed in USP1WT, USP1PIP1, USP1DM1 and USP1PIP1+DM1 on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, technical replicates).D) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA.


Figure 1A . BMDMM were treated as indicated with either 60ng / mL ultra - pure ( UP ) LPS , 50ng / mL human derived recombinant GM - CSF , 40µM WEHD - FMK for 16 hours or 5µM nigericin for the last hour . Supernatants were then analysed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown . B . HoxB8 macrophages were treated with the indicated dilutions of GM - CSF using supernatants from GM - CSF expressing cells . Shown are ELISA analysis of IL - 1β secretion in supernatants and western blots indicating IL - 1β expression in lysates . . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown . C . HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng / mL UP - LPS , 1 % GM - CSF containing supernatant ( approximately 2ng / mL GM - CSF ) , 40µM WEHD . FMK , 0 , 5µM CRT - 0066101 for 16 hours , 5µM nigericin was added for the last hour . Supernatants were analysed by ELISA for IL - 1β . Inlay panels show western blots for levels of IL - 1β expression in the indicated knockouts . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown . D . HoxB8 cells were treated with 60ng / mL of UP - LPS , 1 % GM - CSF supernatant , 50mM KCl , and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL - 1β by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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

Figure 1A. BMDMM were treated as indicated with either 60ng/mL ultra-pure (UP) LPS, 50ng/mL human derived recombinant GM-CSF, 40µM WEHD-FMK for 16 hours or 5µM nigericin for the last hour. Supernatants were then analysed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.B. HoxB8 macrophages were treated with the indicated dilutions of GM-CSF using supernatants from GM-CSF expressing cells. Shown are ELISA analysis of IL-1β secretion in supernatants and western blots indicating IL-1β expression in lysates.. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.C. HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng/mL UP-LPS, 1% GM-CSF containing supernatant (approximately 2ng/mL GM-CSF), 40µM WEHD.FMK, 0,5µM CRT-0066101 for 16 hours, 5µM nigericin was added for the last hour. Supernatants were analysed by ELISA for IL-1β. Inlay panels show western blots for levels of IL-1β expression in the indicated knockouts. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.D. HoxB8 cells were treated with 60ng/mL of UP-LPS, 1% GM-CSF supernatant, 50mM KCl, and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL-1β by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.