( D ) COUP - TFII - OE , in absence of TGFβ1 stimulation , displayed an elongated fibroblast shape with increased αSMA expression , similar to WT treated with TGFβ1 . by confocal in 400x hpf , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA , mean ± SD .

(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 .

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(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.


( D ) WT , COUP - TFII overexpression ( OE ) COUP - TFII knockout ( KO ) were seeded in Seahorse XF - 24 culture microplates . were rendered quiescent in 0 . 5 % FBS DMEM overnight then treated with or without 10 ng / ml TGFβ1 for 24 hours . were incubated in glycolysis stress medium without glucose pyruvate , followed by sequential treatments with glucose ( 10mM ) , oligomycin ( 5μg / ml ) 2 - DG ( 50mM ) . Real - time extracellular acidification rate ( ECAR ) was recorded as baseline ( before glucose ) , rate of glycolysis ( after glucose ) , glycolytic capacity ( after oligomycin ) , glycolytic reserve ( after 2 - DG ) ( n = 10 ) .

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(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 baseline , rate of glycolysis , glycolytic capacity , glycolytic reserve when compared to WT under TGFβ1 stimulation . Without TGFβ1 treatment , there is no difference on baseline glycolysis among WT , OE , KO . COUP - TFII KO significantly decreased glycolytic capacity 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 .

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(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.


( A ) Volcano plot of detected from proteomics of TGFβ  - treated naïve ( WT ) COUP - TFII overexpressing ( COUP - TFII - OE ) . PDK4 expression is significantly decreased in OE ( n = 2 ) .

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(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 PDK4 , both are involved in key steps of FAO . However , overexpression of PGC1α did not abrogate TGFβ1 up - regulated genes in glycolysis αSMA ( n = 6 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

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(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.


( B ) Non - supervised clustering plot indicates substantial clustering of transcripts from each tissue but not of transcripts from compared to controls . PD samples indicated in red .

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(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 in SN samples of PD controls , normalized to beta - actin mRNA . T - p * = 0 . 025 , Data presented as mean ± SD . n = 18 for CT 24 for PD .

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(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.


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

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


( A ) Total circRNAs detected in SN , MTG AMG of both PD control donors , p * = 0 . 02 p * = 0 . 026 p * = 0 . 022 for comparison between PD CT in SN , MTG AMG . T - for total SN vs MTG p * = 2 . 27E - 8 SN vs AMG p * = 5 . 86E - 10 . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control 13 for MTG PD , 10 for SN control 15 for SN PD . box is drawn from Q1 to Q3 with a horizontal drawn in middle to denote median x marks average . Whiskers mark minimum or maximum values . P value was calculated by T - .

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(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 in 3 brain regions is on Alu editing index , representing weighted average editing level across expressed Alu sequences , Wilcoxon , corrected p * = 0 . 038 , 0 . 055 0 . 42 for Amygdala , MTG SN . Wilcoxon between Alu editing index of control PD samples .

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(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 editing on Alu editing index , studied only in Alu elements within circRNA exons their flanking introns , in 3 brain regions , for circRNAs corrected p * = 0 . 025 0 . 040 for AMG MTG p = 0 . 3 for SN . For flanking introns p * = 0 . 02 0 . 040 for AMG MTG p = 0 . 34 for SN , Wilcoxon . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control 13 for MTG PD , 10 for SN control 15 for SN PD . box is drawn from Q1 to Q3 with a horizontal drawn in middle to denote median x marks average . Whiskers mark minimum or maximum values .

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(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 editing circRNA abundance , correlation = - 0 . 37 , Correlation p value = 0 . 0015 .

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(D) Negative correlation between total RNA editing and circRNA abundance, correlation = -0.37, Correlation test p value = 0.0015.


( E ) Adar1 expression in tissues . box is drawn from Q1 to Q3 with a horizontal drawn in middle to denote median x marks average . Whiskers mark minimum or maximum values .

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(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 of control ( green , correlation = 0 . 68 , p - value = 0 . 032 ) PD donors ( blue , correlation = - 0 . 14 p value = 0 . 62 ) .

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(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 ( DEseq2 ) . CircSLC8A1 is marked is red .

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(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.


( B , C ) CircSLC8A1 linear SLC8A1 in SN of control PD brains , T - p = 0 . 025 , p = 0 . 118 respectively .

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(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 SLC8A1 mRNA changes in SN , T - p * = 0 . 025 . n = 18 for CT 24 for PD , x defines mean values .

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(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 in , T - p = 0 . 583 for control p = 0 . 262 for PD . n = 15 16 . . box is drawn from Q1 to Q3 with a horizontal drawn in middle to denote median x marks average .

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(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 linear SLC8A1 mRNA are correlated in control , R2 = 0 . 47 T - p = 0 . 0017 but not in PD samples , R2 = 0 . 016 , p > 0 . 05 . n = 18 for CT 23 for PD .

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(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 in PQ - exposed SH - SY ( normalized to Tubb3 RPL19 ) while other circRNAs remain

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(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 ) of SLC8A1 in PQ - exposed SH - SY , with Tubulin as a loading control , T - p * = 0 . 015 . n = 3 biological replicas for each condition . Data presented as mean ± SD .

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(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.


( A ) qPCR measurements of circSLC8A1 SLC8A1 mRNA after SH - SY treatment of statins or PF LRRK2 inhibitor , T - p * * = 0 . 007 p * * = 0 . 01 for PF - 06447475 p * = 0 . 047 p = * 0 . 0136 for Statins for cirSLC8A1 SLC8A1 mRNA

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(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 ) gel of SLC8A1 Tubulin as a loading control in Statin PF treated neuronal cultures , T - p * = 0 . 027 for Statins p * = 0 . 011 for PF - 06447475 , n = 3 biological replicas for each condition . Data presented as mean ± SD .

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(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 .

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(D) SLC8A1 immunostaining of statin-treated SH-SY cells.


( B ) qPCR from / fractionations of SH - SY , T - p = 0 . 005 for circSLC8A1 , p = 0 . 009 for NEAT1 p = 0 . 009 for Rpl19 . n = 3 biological replicas for each condition .

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(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 of Ago2 - bound , immunoprecipitated ( IP ) / Input values normalized to negative control for a known Ago2 target , circCDR1as circSLC8A1 , T - p = 0 . 029 for circSLC8A1 p = 0 . 001 for circCDR1as , n = 3 biological replicas for each condition .

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(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 - seq , p * * = 0 . 0004 for BMI1 , p * = 0 . 011 for SIRT1 p * = 0 . 005 for AXIN1 .

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(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 treated with Paraquat , T - p * = 0 . 03 , p * = 0 . 02 p * = 0 . 002 for AXIN1 , BM1 SIRT1 respectively , Data presented as mean ± SD with 4 - 6 biological replicas for each condition .

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(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.


A In silico of Sirt7 mRNA expression in different tissues of BXD genetic reference population ( www . genenetwork . org ) . Adr : Adrenal 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 mRNA , Skin : Skin Back mRNA , Sn : Sciatic Nerve mRNA , Spl : Spleen mRNA , Tes : Testis mRNA , Thy : Thymus mRNA , Ton : Tongue mRNA , Tra : Trachea mRNA , Ute : Uterus mRNA .

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A 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 Slc12a7 that codifies KCC4 in mouse BxD genetic reference population ( www . genenetwork . org ) .

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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 NAM 10 mM for 4 h . Lysates were immunoprecipitated with an anti - KCC4 antibody 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 .

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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 indicated times , KCC4 ( upper panel ) , activity ( lower panel ) were analyzed by SDS - PAGE / immunoblotting or Cl - - dependent 86Rb + uptake under hypotonic conditions .

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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 indicated times , KCC4 were analyzed by SDS - PAGE / immunoblotting .

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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 incubated with vehicle ( V ) , NAD + or NAM for 48 h , KCC4 ( upper pannel ) , activity ( lower pannel ) were analyzed as in D .

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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.


A , B KCC4 activity ( A ) , ( B ) were evaluated in Xenopus oocytes expressing SIRT7 KCC4 that were incubated for indicated times with NAD + . Non - injected oocytes ( UI ) were used as a control .

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A, 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 coinjected with either wild - type ( SIRT7 WT ) or catalytically inactive SIRT7 ( SIRT7 H188Y ) for 48 h .

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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 then incubated with vehicle ( V ) , NAD + or NAM for 24 h , KCC4 SIRT7 were analyzed by SDS - PAGE / immunoblotting .

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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 without SIRT7 for 48 h then incubated with vehicle ( V ) or NAM for 4 h . After lysis , KCC4 was immunoprecipitated using an anti - KCC4 antibody , acetylation status of KCC4 was determined by SDS - PAGE / Western blotting using anti - acetyl lysine antibody .

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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 SIRT7 were transfected into HEK293 treated for indicated times with NAD + ( F ) or NAM ( G ) then analyzed by SDS - PAGE / immunoblotting using an anti - KCC4 or anti - SIRT7 antibody . Non - transfected ( UT ) were used as a control .

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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 SIRT7 were transfected into HEK293 treated for indicated times with NAD + NAM . After lysis , KCC4 immunoprecipitation using an anti - FLAG antibody was performed , interaction with SIRT7 was determined by SDS - PAGE / Western blotting .

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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 expressing KCC4 were incubated with NAD + 200 μM cycloheximide ( CHX ) for 0 , 3 , 6 9 h . KCC4 are shown at indicated times in transfected with a scramble shRNA ( + SIRT7 ) or a shRNA against SIRT7 ( - SIRT7 ) .

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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).


A KCC4 acetylation at indicated pH in HEK293 transfected with KCC4 . After lysis , KCC4 was immunoprecipitated using an anti - FLAG antibody , then analyzed by SDS - PAGE / Western blotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - transfected ( UT ) were used as a control .

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A 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 were transfected with KCC4 together with SIRT7 with a scramble shRNA ( - ) or a shRNA against SIRT7 ( + ) , KCC4 activity level were determined at indicated pH . SIRT7 expression in these conditions is presented in C .

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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 SIRT7 at different pH values in HEK293 . At a lower pH , SIRT7 appears to translocate to cytoplasm .

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C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells. At a lower pH, SIRT7 appears to translocate to the cytoplasm.


A , B Sirt7 mRNA ( A ) ( 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 methods ( Spirli et al , 2019 ) .

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A, 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 ATPase in kidneys of C57BL6 mice with metabolic acidosis ( SIRT7 , red ; ATPase , green ; DAPI , Blue ) . Scale bar : 20 μm . Controls of specificity of antibodies using kidney of SIRT7 - deficient mice are provided in Appendix Fig S4 .

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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 SIRT7 in kidneys from control mice or mice with metabolic acidosis . Values are mean ± .

D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis. Values are the mean ± SEM.


A KCC4 were determined in kidneys of wild - type ( SIRT7 + / + ) SIRT7 - deficient mice ( SIRT7 - / - ) . SIRT7 expression are absent in kidneys of SIRT7 - deficient mice .

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A 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 , acetylation status of KCC4 , interaction with SIRT7 in kidneys of SIRT7 + / + SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis .

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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 H - ATPase in kidneys of SIRT7 + / + SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis ( NH4Cl ) . ( KCC4 , red ; H - ATPase , green ) . Scale bar : 20 μm . was performed automatically using Gen5 software to report of KCC4 + among H - ATPase + ( lower panel ) .

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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 NKCC2 in kidney of SIRT7 + / + SIRT7 - / - mice under basal conditions .

D NCC and NKCC2 protein levels in the kidney of SIRT7+/+ and SIRT7-/- mice under basal conditions.


HeLa lines were treated with 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 ) .

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HeLa 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 lines were treated with 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 ) .

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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 lines were treated with 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 ) .

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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 lines were treated with 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 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 lines were treated with Mcl - 1 - inhibitor S63845 for 72 h . Cytokines were measured by ELISA . ( E ) HeLa CTRL Bax / Bak - deficient were treated with Mcl - 1 - inhibitor S63845 . Concentrations of IL - 6 in supernatants are shown ( CTRL , n = 8 ; Bax / Bak , n = 7 ) .

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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 or 1205Lu melanoma over - expressing Bcl - XL were treated with ABT - 737 . Concentrations of IL - 6 in supernatants were measured after 48 h ( n = 5 ) .

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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 or 1205Lu melanoma over - expressing mouse Bcl - XL were treated with various concentrations of ABT - 737 . To some aliquots caspase - inhibitor zVAD - fmk ( 50 μM ) was added . Relative numbers of apoptotic were determined as in EV 1B after 48 h . Data are means / of at least three independent experiments . ( 1205Lu , n = 4 ; 1205Lu 5 µM ABT - 737 , n = 3 ; 1205Lu / Bcl - XL 1205Lu + zVAD , n = 3 ) .

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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 untreated or treated with tamoxifen ( 100 nM , 24 h ) were stained with Picogreen ( 3 µl / ml ) to detect mitochondrial . Scale bare , 10

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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 µm


I HeLa CTRL HeLa - UL12 . 5 were treated with ABT - 737 ( 10µM ) . of IL - 6 in supernatant were measured after 72h ( n = 3 ) .

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I 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).


A , caspase - 3 - reporter ( labelled DEVD ) control ( DEVG ; see methods ) were infected 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 ( IAV ; MOI = 1 , 16h ) ; Herpes simplex 1 ( HSV - 1 ; MOI = 1 , 16h ) . Staurosporine treated ( 1 μM , 3h ) were used as a positive control . Percentages of positive for caspase - 3 - activation ( FRET - loss ) were determined . Data are means / of three independent experiments . ( n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, 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 were infected with modified vaccinia Ankara ( MVA ) ( MOI = 10 ) . caspase - inhibitor zVAD - fmk was used at 100µM ; were analysed 16 h post - infection .

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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 were infected with influenza A ( IAV ) ( MOI = 1 ; was 16 h post - infection ) . D , detection of viral M2 was used as infection control .

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 were infected with Herpes simplex 1 ( HSV - 1 ) ( MOI = 1 ; was 16 h post - infection ) .

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F, G Cells were infected with Herpes simplex virus 1 (HSV-1) (MOI=1; analysis was 16 h post-infection).


H were infected with Chlamydia trachomatis ( Ctr ) ( MOI = 1 ; was 30 h post - infection ) .

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H Cells were infected with Chlamydia trachomatis (Ctr) (MOI=1; analysis was 30 h post-infection).


I were infected with Salmonella Typhimurium ( Sty ) ( MOI = 50 ; was 24 h post - infection ) .

I Cells were infected with Salmonella Typhimurium (Sty) (MOI=50; analysis was 24 h post-infection).


J were infected with protozoan Toxoplasma gondii ( MOI = 5 ; was 16 h post - infection ) .

J Cells were infected with the protozoan parasite Toxoplasma gondii (MOI=5; analysis was 16 h post-infection).


A Infection with MVA ( MOI = 10 for 16 h ; IL - 6 ) .

A Infection with MVA (MOI=10 for 16 h; IL-6).


B infection with HSV - 1 ( MOI = 1 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 -

Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; IL-6


Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; , IL - 8 ) .

Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; , IL-8).


Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; IL -

Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; IL-6


Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; CXCL - 1 ) .

Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; CXCL-1).


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; IL -

Infection with Toxoplasma gondii (MOI=5 for 16 h; IL-6


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; , IL -

Infection with Toxoplasma gondii (MOI=5 for 16 h; , IL-8


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; CXCL -

Infection with Toxoplasma gondii (MOI=5 for 16 h; CXCL-1


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; FGF - 2 ) .

Infection with Toxoplasma gondii (MOI=5 for 16 h; FGF-2).


( A ) HeLa 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 ) . were analyzed by flow cytometry at indicated time points . were gated on GFP - positive ( infected ) host population , mean was recorded . Data are means / of six experiments . ( A ) Growth of C . trachomatis was measured in HeLa as increase in GFP - between 18 38 h . data show means / of six independent experiments ( genotype effect ( significance of difference of GFP - expression between lines overall , p = 0 . 08 ) . Red indicates fold change calculation of different time points lines ( significance tested ( one sample t - ) for each time point for a difference between Bax / Bak vs CTRL ) .

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(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 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 ) . were analyzed by flow cytometry at indicated time points . were gated on GFP - positive ( infected ) host population , mean was recorded . Data are means / of 4 - 5 experiments . ( B ) Growth of C . trachomatis was measured in AGS as increase in GFP - . data show means / of 4 to 5 independent experiments ( genotype effect : p < 0 . 0001 ; time - genotype interaction : p < 0 . 0001 ) ( 12 h 38 h , n = 4 ; 18 h , n = 5 ) .

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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 culture ( expressed as chlamydial in 50 ng of total , determined by quantitative PCR ) . * , p < 0 . 05 between control mutant . ( n = 3 )

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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 were infected with Salmonella Typhimurium ( MOI = 50 ) . Extracellular were killed using non - permeable antibiotic . At indicated time points were lysed , aliquots were plated on agar plates . Recovered colony forming units were calculated . Data are means / of four independent experiments . Significant genotype effects were found between mutant control ( Bax / Bak p < 0 . 0001 ; Bcl - XL p 0 . 0001 ) .

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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 were infected with Salmonella Typhimurium ( MOI = 50 ) . CFU / well were calculated as in ( D ) . n = 3 .

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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.


A Actin expression shown by western blotting for strains expressing S . cerevisiae ' s actin from various coding sequences , with tubulin ( Tub1p ) as a loading control . B of actin expression , 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 ] Sc [ At ] ; 2 biological replicates with n / 2 technical replicates each ) . * P < 0 . 05 ( Brown - Forsythe Welch ANOVA , with Dunnett ' s T3 multiple comparisons ) .

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A 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 patch - cable balance compared to S . cerevisiae haploid ( value is 0 in S . cerevisiae ' s , 1 when contain only actin patches - 1 when contain only cables ) . Data are presented as mean + / - SD ( n = 30 for conditions ) . * * * P < 0 . 001 ( Brown - Forsythe Welch ANOVA , with Dunnett ' s T3 multiple comparisons ) .

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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).


E Phalloidin of F - actin organization . are maximum projections of 3D stacks contrasts were adapted due to fact that phalloidin labeling had a very different efficiency depending on actin ortholog expressed . of Sc Sc _ NI are reproduced from Fig 2E . Scale bar : 3 µm .

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E 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 conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe Welch ANOVA , with Dunnett ' s T3 multiple comparisons ) .

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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 organization of actin cytoskeleton . were stained with phalloidin after 30 min incubation with CK - 666 . are maximum projections of 3D stacks . Scale bar : 3 µm J of actin patch resistance to CK - 666 treatment . Bar graphs represent of 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 ) .

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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).


A ( Top ) Snapshots of actin networks assembled from three different actins sources : Act _ Sc , Act _ N2 Act _ Ca . ( Bottom left ) of actin 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 n = 47 for Act _ Ca linear ) . * * P < 0 . 01 , * * * P < 0 . 001 ( Kruskal - Wallis , with multiple comparisons ) . ( Bottom right ) In vitro actin network deviation indexes .

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A (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 presence of 600 nM Alexa - 488 - labeled ADF / cofilin ( replacement of unlabeled ADF / cofilin ) . ( Right ) of ADF / cofilin ' s 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 n = 27 for Act _ Ca linear ) . * * * P < 0 . 001 ( Kruskal - Wallis , with multiple comparisons ) .

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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 presence 1 µM Alexa 488 - tropomyosin . ( Right ) of tropomyosin ' s 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 n = 27 for Act _ Ca linear ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe Welch ANOVA , with Dunnett ' s T3 multiple comparisons ) .

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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).


C Phalloidin depicting F - actin organization . are maximum projections of 3D stacks . Scale bar : 3 µm . D In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe Welch ANOVA , with Dunnett ' s T3 multiple comparisons ) . E Polarity Indexes . Data are presented as mean + / - SD ( n = 30 for conditions ) . * * * P < 0 . 001 ( Kruskal - Wallis , with multiple comparisons ) .

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C 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 organization of actin cytoskeleton . were stained with phalloidin after 30 min incubation with CK - 666 . are maximum projections of 3D stacks . Scale bar : 3 µm . G In vivo actin network deviation indexes of treated with DMSO or CK - 666 . Data are presented as mean + / - SD ( n = 30 for conditions ) . * * * P < 0 . 001 ( Brown - Forsythe Welch ANOVA , with Dunnett ' s T3 multiple comparisons ) .

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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).


( C ) 3 . 1 Å 2Fo - Fc map ( 1 . 2 σ ) around of final refined ATPγS - MjMR - structure .

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(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 3 . 1 Å ATPγS - MjMR - structure showing Mre11 nuclease , capping , C - terminal three helix - bundle domain ( pink ) , Rad50 molecules ( green blue ) , ( yellow orange ) . ATPγS is shown in spheres . See Movie EV 1 for movement of .

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(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.


( A ) A ribbon diagram showing overall recognition by ATPγS - Rad50 dimer .

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(A) A ribbon diagram showing the overall DNA recognition by the ATPγS-Rad50 dimer.


( B ) Close - up view of interaction between internal segment ( major minor grooves ) of strand edge of lobe I of Rad50A ( green ) . Loop α1 - α2 wedge faces minor groove between 10th 13 ' th phosphates of template ( orange ) non - template ( yellow ) strands , respectively . ( C ) Close - up view of interaction between strand α1 - α2 loop in an orientation different from Fig 2B .

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(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.


( B , C ) binding for Interactions between WT or four MjMRcd mutants a closed circular dsDNA ( φx174RFII ) in absence ( B ) or presence ( C ) of AMP - PNP / Mg2 + . Each sample was incubated with φx174RFII dsDNA ( 3 . 5 nM ) for 30 min on 4 ° C . following molar of : 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 with .

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(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 WT or five full - TmMR mutants with φx174RFII were examined in absence ( D ) or presence of AMP - PNP / Mg2 + ( E ) . Reaction conditions are same as those in Fig 3B .

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(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 ) - bound MjMRcd ( 7 , 8 ) towards a hairpin . Each sample was incubated with a hairpin ( 10 nM ) in a 1 : 10 or 1 : 20 molar ( : ) for 30 min on 55 ° C . error bars for quantified values of uncleaved substrates on right panel are calculated from standard deviation from three repetitions of each experiment .

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(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 ) - bound ( lane 7 , 8 ) full - TmMR towards a hairpin .

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(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.


( A ) Superposition of ATPγS - MjMR - ( green ) apo - MjMR ( 3AV0 , magenta ) structures . Alignment was done using their NBDs ( 1 . 3 Å rmsd in positions of 311 Cα atoms ) . coiled - coils of - bound Rad50 molecules are shifted ( arrow ) to more parallel orientation . entire structures can be superimposed with a 1 . 9 Å rmsd in 692 Cα positions .

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(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 local structures between apo ( B ) - bound MjMR ( C ) . Upon binding , helix α6 translates to backbone , resulting in tighter packing of Leu155 Leu156 against a hydrophobic pocket formed by lobe I II . Arrows indicate direction of helix movement by binding . See Fig EV5 for a close - up view .

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(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.


( A ) Stereo view of superposition of ADP - MjRad50 ( 3AUX ) ( black ) onto ATPγS - MjMR - ( green blue ) by aligning their lobe II domains . α1 - α2 loop that could collide with parts of molecule is indicated . ( B ) Aligning lobe II domains of two structures in Fig 5A shows that rotation of lobe I opens gate for Mre11 , which could allow access of unwound . Color schemes are same as those in Fig 5A .

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(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 ) - unwinding activities of TmMR . Lane 2 , no ; lanes 3 4 , no ATP ; lanes 5 6 , with ATPγS ; lanes 7 8 , with ATP . : was 1 : 2 for lanes 3 , 5 , 7 , 1 : 5 for lanes 4 , 6 , 8 . Plotted data represent mean of three experiments , with error bars indicating one standard deviation . A graph of quantified data is shown in lower panel .

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(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 ATP - dependent unwinding activities of WT mutant TmMR . Lane 2 , no ; lane 3 4 , WT TmMR ; lane 5 6 , Rad50 ( Δ54 - 56 ) mutant ; lane 7 8 , R87E mutant ; lane 9 10 , K95E mutant ; lane 11 12 , R94E / K95E mutant ; lane 13 14 , K115E mutant . For each TmMR , reaction contains 1 : 2 or 1 : 5 of : .

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(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.


( A ) Endonuclease activities of TmMR WT mutant towards a duplex in absence ( left panel ) or presence of ATP ( right panel ) . Major products are marked with dots . 5 ' - 32P - label is indicated by a star . See Appendix Fig S4A S4B for quantified values .

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(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 ) of a bubble binding by WT or mutant TmMR in presence or absence of AMP - PNP . See Appendix Fig S3E S3F for original data .

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(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 WT mutant TmMR towards a bubble molecule in absence ( left panel ) or presence of ATP ( right panel ) . See Appendix Fig S4C S4D for quantified values .

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(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 ) effects of single or multiple point mutations in - contacting or neighboring residues of S . cerevisiae rad50 on sensitivity to chemical drugs . Sensitivities of rad50 expressing mutants towards indicated concentrations of CPT , PHL , MMS were examined . ( E ) effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for quantified values .

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(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 ) effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for quantified values .

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(E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


B ) Single point activity assays of USP1WT deletion mutants on Ub - Rho show signifi cantly increased activity in mutants where insert L1 L3 are both deleted .

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B) 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 deletion mutants ( ± UAF1 ) on Ub - Rho show loss in hyper - activation of USP1ΔL1L3 upon addition of UAF1 .

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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 of USP1 deletion mutants against ubiquitin - rhodamine ( Ub - Rho ) shows that USP1ΔL1L3 has significantly higher activity compared to WT other mutants ( n = 2 , biological replicates ) .

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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 of USP1 deletion mutants ( + UAF1 ) against Ub - Rho shows that deletions mutants have similar catalytic activity ( n = 2 , biological replicates ) .

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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 USP1ΔL1L3 on a peptide substrate i . e . LRGG - AMC ( 100µM ) . USP1ΔL1L3 cleaves peptide substrate more efficiently compared to USP1WT ( n = 2 , technical 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 USP1ΔL1L3 ( + UAF1 ) on a peptide substrate i . e . LRGG - AMC ( 100µM ) . Addition of UAF1 allows cleavage of LRGG - AMC by both USP1WT USP1ΔL1L3 ( n = 2 , technical replicates ) .

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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).


A ) of gel - activity assays where activity of USP1WT deletion mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . USP1ΔL1 mutant has reduced activity on PCNA - UbTAMRA compared to other USP1 variants tested ( n = 2 , biological replicates ) .

A) 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 ) of gel - activity assays where activity of USP1 PIP mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . Both PIP mutants have reduced activity on PCNA - Ub compared to USP1WT ( n = 2 , biological replicates ) .

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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 binding assays of USP1 mutants ( + UAF1 ) with PCNA - UbTAMRA show reduced binding of USP1ΔL1 USP1PIP1 compared to USP1WT ( n = 2 , technical replicates ) .

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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 ) of gel - activity assays where activity of USP1WT + UAF1 on PCNA - UbWT is compared with PCNA - UbPIM1 shows that PCNA - UbWT is much faster compared to PCNA - UbPIM1 ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) )

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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 USP1PIP1 ( + UAF1 ) on PCNA - UbWT PCNA - UbPIM1 in gel - assays shows that activity of USP1WT is severely reduced by PIM1 mutation in PCNA , whereas USP1PIP1 activity is not further affected indicating that PIP1 PIM mutation affect same interaction ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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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)).


A ) Schematic representation of loading purification of PCNA - Ub on nicked circular . - loaded PCNA - Ub elutes in void of SEC column this sample is collected used for studying activity of USP1 on - loaded PCNA - Ub .

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A) 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 of USP1 + UAF1 with without ( 65bp dsDNA ) shows that binding alone has no effect on USP1 activity ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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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 showing increased activity of USP1 - UAF1 on - loaded PCNA - Ub compared to PCNA - Ub ( - ) PCNA - Ub ( + , + RFC not ATP ) .

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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 ) of gel - activity assays showing enhanced activity of USP1 + UAF1 on - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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 ) of gel - activity assays showing enhanced activity of USP1 on - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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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)).


A ) Gel of USP1 - UAF1 activity on PCNA - Ub ( loaded free ) for three different of enzyme substrate .

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A) Gel based quantification of USP1-UAF1 activity on PCNA-Ub (DNA loaded and free) for three different concentration of enzyme and substrate.


B ) FP activity assays on free PCNA - Ub ( three concentrations of USP1 PCNA - Ub ) activity of USP1 on increasing concentrations of Ub - Rho .

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.


A ) Comparison of USP1WT deletion mutants ( + UAF1 ) for activity on - loaded PCNA - Ub ( solid lines ) free PCNA - Ub ( dashed lines ) shows no increase in USP1ΔL1 activity on loaded substrate . Left panel : of gel - activity assays showing of PCNA - Ub at mentioned time points ( n = 2 , biological replicates ) , Right panel : of activation fold observed in USP1WT mutants on - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , biological replicates ) .

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A) 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 alignment of USP1 insert L1 which highlights binding region conservation of positively charged residues across species . Three groups of positively charged residues ( * * * ) are mutated separately tested for binding to double stranded ( 65bp ) using SPR which shows importance of this region for binding of USP1 .

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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 USP1PIP1 + DM1 ( + UAF1 ) activity on - loaded PCNA - Ub ( solid lines ) free PCNA - Ub ( dashed lines ) shows reduced increase in activity of these mutants compared to USP1WT . Left panel : of gel activity assays showing of PCNA - Ub at mentioned time points ( n = 2 , technical replicates ) , Right panel : of activation fold observed in USP1WT , USP1PIP1 , USP1DM1 USP1PIP1 + DM1 on - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , technical replicates ) .

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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 binding experiment shows that USP1PIP1 USP1WT has similar binding capability while USP1DM1 has lost its ability to bind .

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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.


A . 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 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 panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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A. 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 indicated dilutions of GM - CSF using supernatants from GM - CSF expressing . Shown are ELISA of IL - 1β secretion in supernatants western blots indicating IL - 1β expression in lysates . . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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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 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 last hour . Supernatants were analysed by ELISA for IL - 1β . Inlay panels show western blots for of IL - 1β expression in indicated knockouts . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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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 were treated with 60ng / mL of UP - LPS , 1 % GM - CSF supernatant , 50mM KCl , 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 panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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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.


A . BMDMM ( left panel ) HoxB8 macrophages ( right panel ) were treated as indicated with either LPS or LPS + GM - CSF . were harvested extracted analysed by western blot for of NRF2 . Shown is densitometric of Nrf2 level from three independent experiments . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For western blot , significance was calculated using unpaired t - . p values are shown .

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A. BMDMM (left panel) and HoxB8 macrophages (right panel) were treated as indicated with either LPS or LPS+ GM-CSF. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2. Shown is the densitometric quantification of Nrf2 protein level from three independent experiments. 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. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


B . HoxB8 macrophages were treated as in A . of IL - 1β in supernatant were analysed by ELISA Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For western blot , significance was calculated using unpaired t - . p values are shown .

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B. HoxB8 macrophages were treated as in A. and levels of IL-1β in the supernatant were analysed 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. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


C . HoxB8 macrophages were treated for 12 hours with LPS or LPS + GM - CSF extracted analysed by qPCR for indicated targets . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For western blot , significance was calculated using unpaired t - . p values are shown .

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C. HoxB8 macrophages were treated for 12 hours with LPS or LPS+GM-CSF and RNA extracted and analysed by qPCR for the indicated targets. 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. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


D . Wild type Tnf - / - HoxB8 were treated as indicated . were harvested extracted analysed by western blot for of NRF2 HMOX1 . For wild type , MitoTEMPO ( 50µM ) was also added as indicated . Shown are densitometric quantifications of NRF2 HMOX1 of three independent experiments ( right panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For western blot , significance was calculated using unpaired t - . p values are shown .

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D. Wild type and Tnf-/- HoxB8 cells were treated as indicated. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1. For wild type cells, MitoTEMPO (50µM) was also added as indicated. Shown are the densitometric quantifications of NRF2 and HMOX1 protein levels of three independent experiments (right panel). 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. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


E . HoxB8 of indicated genotypes were treated with LPS for 8 hours followed by with mitoSOX red . signal was analyzed using flow cytometry . Shown are average geometric mean of mitoSOX red . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For western blot , significance was calculated using unpaired t - . p values are shown .

E. HoxB8 cells of the indicated genotypes were treated with LPS for 8 hours followed by staining with mitoSOX red. The fluorescent signal was analyzed using flow cytometry. Shown are average geometric mean fluorescence intensity of mitoSOX red. 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. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


F . Tnf - / - HoxB8 macrophages were treated with LPS with or without 50ng / ml recombinant TNF for 16 hours . were harvested extracted analysed by western blot for of NRF2 HMOX1 ( upper panel ) . Shown are densitometric quantifications of indicated of three independent experiments ( lower panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For western blot , significance was calculated using unpaired t - . p values are shown .

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F. Tnf-/- HoxB8 macrophages were treated with LPS with or without 50ng/ml recombinant TNF for 16 hours. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 (upper panel). Shown are the densitometric quantifications of indicated protein levels of three independent experiments (lower panel). 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. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


A . HoxB8 macrophages were seeded in an 8 - well Ibidi chamber treated as indicated for 12 hours . were fixed stained for ASC ( Red ) with DAPI ( blue ) . For nigericin treatments nigericin was added for only 30 minutes . Example containing ASC Specks are shown with arrows . Scale bars represent 50µM . Data information : Microscopy pictures are representative from 3 biological replicates .

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A. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated as indicated for 12 hours. Cells were fixed and stained for ASC (Red) and with DAPI (blue). For nigericin treatments nigericin was added for only 30 minutes. Example cells containing ASC Specks are shown with white arrows. Scale bars represent 50µM. Data information: Microscopy pictures are representative images from 3 biological replicates.


B . HoxB8 were treated as indicated with LPS or LPS + GM - CSF . cross linker DSS was added to as described in methods . Samples were analysed by western blot for ASC oligomerization . Black vertical lines indicate that non - relevant lanes were removed during figure preparation but samples were run on same gel . Data information : westerns are representative from 3 biological replicates .

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B. HoxB8 cells were treated as indicated with LPS or LPS+GM-CSF. The cross linker DSS was added to the cells as described in methods. Samples were analysed by western blot for ASC oligomerization. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel. Data information: westerns are representative images from 3 biological replicates.


C . Wild type caspase - 1 - / - HoxB8 macrophages were treated as indicated for 16 hours . lysates were made analysed by western blot for of caspase - 1 gasdermin D cleavage . Data information : westerns are representative from 3 biological replicates .

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C. Wild type and caspase-1-/- HoxB8 macrophages were treated as indicated for 16 hours. Cell lysates were made and analysed by western blot for levels of caspase-1 and gasdermin D cleavage. Data information: westerns are representative images from 3 biological replicates.