Quantitative RT - PCR of IL - 6mRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP , and Cx3cr1GFP / GFP ApoE − / − peritoneal macrophages cultured for 24 h with CX3CL1 ( n = 5 per group , one - way ANOVA / Bonferroni post hoc multi - comparison tests : C57BL / 6J versus Cx3cr1GFP / GFP * P < 0 . 0001 and Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P < 0 . 0001 . Mann - Whitney U - test : C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0079 and Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P = 0 . 0079 . The experiment was repeated twice with similar results . ) .

Quantitative RT-PCR of IL-6mRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP, and Cx3cr1GFP/GFP ApoE−/− peritoneal macrophages cultured for 24 h with CX3CL1 (n = 5 per group, one-way ANOVA/Bonferroni post hoc multi-comparison tests: C57BL/6J versus Cx3cr1GFP/GFP *P < 0.0001 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P < 0.0001. Mann-Whitney U-test: C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0079 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P = 0.0079. The experiment was repeated twice with similar results.).


Orthogonal and lateral Z - stack projection of IL - 6 ( D , red ) , IBA - 1 + ( E , green ) , and phalloidin ( E , blue ) of IBA + mononuclear phagocytes adjacent to the phalloidin + RPE of a retinal flatmount from a 12 - month - old Cx3cr1GFP / GFP mouse . ( Representative of three independent experiments , immunostainings omitting the primary antibody served as negative controls ) .

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

Orthogonal and lateral Z-stack projection of IL-6 (D, red), IBA-1+ (E, green), and phalloidin (E, blue) of IBA+ mononuclear phagocytes adjacent to the phalloidin+ RPE of a retinal flatmount from a 12-month-old Cx3cr1GFP/GFP mouse. (Representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL / 6J CFSE + macrophages into C57BL / 6J with or without IL - 6 ( n = 15 - 20 / group ; calculated intraocular concentrations of 5 ng / ml . Mann - Whitney U - test : * P < 0 . 0001 ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFPCFSE + Mϕs into C57BL / 6J with control ( IgG , n = 16 ) or anti - IL - 6 antibody ( aIL - 6 Ab , n = 8 ; calculated intraocular concentrations 5 μg / ml ; per group . Mann - Whitney U - test : * P = 0 . 0036 ) .

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

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6J CFSE+ macrophages into C57BL/6J with or without IL-6 (n = 15-20/group; calculated intraocular concentrations of 5 ng/ml. Mann-Whitney U-test: *P < 0.0001).Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFPCFSE+Mϕs into C57BL/6J with control (IgG, n = 16) or anti-IL-6 antibody (aIL-6 Ab, n = 8; calculated intraocular concentrations 5 μg/ml; per group. Mann-Whitney U-test: *P = 0.0036).


7 day laser - injured IBA - 1 ( green ) and CD102 ( red ) double - stained RPE flatmounts of control IgG - ( I ) and anti - IL - 6 - treated ( J ) Cx3cr1GFP / GFP mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes / impact localized on the lesion surrounding RPE of Cx3cr1GFP / GFP mice treated with control IgG , IL - 6 - , or CD14 - blocking antibodies ( calculated intraocular concentration 5 μg / ml ; n = 13 - 14 / group , one - way ANOVA / Dunnett ' s post hoc tests of IgG versus any other group * P < 0 . 001 . Mann - Whitney U - test IgG versus anti - IL - 6 * P = 0 . 0021 ; IgG versus anti CD14 * P = 0 . 0028 ) .

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

7 day laser-injured IBA-1 (green) and CD102 (red) double-stained RPE flatmounts of control IgG- (I) and anti-IL-6-treated (J) Cx3cr1GFP/GFP mice.Quantification of subretinal IBA-1+ mononuclear phagocytes/impact localized on the lesion surrounding RPE of Cx3cr1GFP/GFP mice treated with control IgG, IL-6-, or CD14-blocking antibodies (calculated intraocular concentration 5 μg/ml; n = 13-14/group, one-way ANOVA/Dunnett's post hoc tests of IgG versus any other group *P < 0.001. Mann-Whitney U-test IgG versus anti-IL-6 *P = 0.0021; IgG versus anti CD14*P = 0.0028).


A Neonatal rat ventricular myocytes ( NRVMs ) were infected with the 3xMEF2 - Luc reporter , serum starved for 20 hours and stimulated for 24 hours with different agonists : 100 nM endothelin - 1 ( ET1 ) , 1 μM sphingosine - 1 - phosphate ( S1P ) , 10 μM lisophosphatidic acid ( LPA ) , 1 μM WIN55 , 212 - 2 ( WIN55 , cannabinoid receptor agonist ) , 1 μM isoproterenol ( ISO , β - adrenergic receptor agonist ) , 100 nM angiotensin II ( AngII ) , 10 μM prostaglandin E1 ( PGE1 ) , 10 μM prostaglandin E2 ( PGE2 ) or 100 nM prostanoid F receptor agonist fluprostenol ( Flupro ) , 100 nM treprostinil ( Trepro , prostacyclin receptor agonist ) Data information : values are mean ± s . e . m . In ( A ) , the experiment was performed in triplicates , similar results were obtained in 3 different experiments . Student ' s two - tailed t - test , * , P < 0 . 05 vs . control ( ET1 , P = 0 . 0013 ; S1P , P = 0 . 0144 ; LPA , P = 0 . 026 ; WIN55 , P = 0 . 6205 ; ISO = 0 . 0399 ; AngII , P = 0 . 5812 ; PGE1 , P = 0 . 001 ; PGE2 , P < 0 . 0001 ; Flupro , P = 0 . 0846 ; Trepro , P = 0 . 1958 )

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A Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist) Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958)


B PGE2 induces the expression of MEF2 target genes . NRVMs were serum - starved for 24 hours , then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information : values are mean ± s . e . m In ( B ) , n = 5 , technical replicates , Student ' s two - tailed t - test , * , P < 0 . 05 vs . control ( Nur77 , P = 0 . 0148 ; Myomaxin , P = 0 . 0417 ; Adamts1 , P = 0 . 0112 ; BNP , P = 0 . 0065 )

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B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information: values are mean±s.e.m In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065)


C Correlation between MEF2 activation and NRVM hypertrophy . MEF2 activity was measured as in ( A ) upon increasing concentration of PGE2 . Cell size of NRVMs was determined after 48 - hour stimulation with the indicated concentrations of PGE2 . Left : representative images of α - actinin stained NRVMs stimulated with DMSO , 300 nM PGE2 , or 1 nM ET1 . Scale bar is 10 µm Data information : values are mean ± s . e . m In ( C ) , NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2 , MEF2 - activity was parallel assessed from 6 technical replicates . Correlation was statistically analyzed by calculating Pearson correlation coefficient ( Pearson r = 0 . 9626 , P = 0 . 0021 )

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C Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-hour stimulation with the indicated concentrations of PGE2. Left: representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 µm Data information: values are mean±s.e.m In (C), NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2, MEF2-activity was parallel assessed from 6 technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r=0.9626, P=0.0021)


D , PGE2 activates MEF2 via EP3 receptor . NRVMs were infected with the 3xMEF2 - Luc reporter and serum - starved for 20 hours . The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists : AH6809 ( 10 μM , EP1 - and EP2 - antagonist ) or 798106 ( 200 nM , EP3 - antagonist ) or L161 , 982 ( 2 μM , EP4 - antagonist ) Data information : values are mean ± s . e . m In ( D ) , n = 3 , independent experiments , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , AH6809 , P = 0 . 1092 ; L798106 , P = 0 . 0002 ; L161 , 982 , P = 0 . 5579 ) . The exact n and P values can be also found in the Source Data excel file for Figure

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D, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist) Data information: values are mean±s.e.m In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1


B - D ( B , D ) NRVMs were infected with the 3xMEF2 - Luciferase reporter and with recombinant adenoviruses encoding EGFP ( AdGFP ) , RGS16 , RGS2 , p63ΔN , RGS - LSCII , or Gαt , as indicated . The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours . ( C ) NRVMs were infected with the 3xMEF2 - Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 ( 100 μM ) for 20 minutes or the Gi / o - protein inhibitor pertussis toxin ( PTX , 100 ng / ml ) for 20 hours in serum - starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours . Data information : In ( B - D ) , n = 3 , independent experiments , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , RGS16 , P < 0 . 0001 ; RGS2 , P = 0 . 1725 ; p63ΔN , P = 0 . 0666 ; RGS - LSCII , P = 0 . 0818 , SQ22536 , P = 0 , 385 ; PTX , P < 0 . 0001 ; Gαt , P < 0 , 0001 ) , values are mean ± s . e . m . The exact n and P values can be also found in the Source Data excel file for Figure 2

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

B-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours. Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2


A , B NRVMs were infected with recombinant adenovirus encoding Flag - HDAC5 ( A ) or not infected ( B ) and serum starved for 20 hours . The cells were pretreated with the PKC - inhibitor bisindolylmaleimide I ( 2 μM ) or the PKD - inhibitor BPKDi ( 3 μM ) for 20 minutes and stimulated with 100 nM endothelin - 1 ( ET1 ) or 100 μM phenylephrine ( PE ) or 1 μM PGE2 for 4 hours , as indicated . HDAC phosphorylation was detected by immunoblotting with the anti - HDAC5 phospho - Ser - 498 antibody , phosphorylation of PKD was assessed using anti - phospho - PKD antibodies ( Ser - 744 / Ser - 748 and Ser - 916 ) , representative images are shown from 3 independent experiments

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O

A,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments


C NRVMs were infected with the 3xMEF2 - Luc reporter and serum - starved for 20 hours . The cells were pretreated with the PKD - inhibitor BPKDi ( 3 μM ) or the CamKII - inhibitor AIP ( 1 μM ) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours . N = 3 , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , BPKDi , P < 0 . 0001 ; AIP , P = 0 . 2197 ) , values are mean ± s . e . m . The exact n and P values can be also found in the Source Data excel file for Figure

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C NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3


A NRVMs were serum - starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2 . Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information : In ( A - C ) representative images from at least 3 independent experiments are shown

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A NRVMs were serum-starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2. Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information: In (A-C) representative images from at least 3 independent experiments are shown


B NRVMs were infected with recombinant adenoviruses encoding EGFP ( AdGFP ) or RGS16 24 hours prior to stimulation , then Rac1 activity was measure Data information : In ( A - C ) representative images from at least 3 independent experiments are shown . In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS16 , P = 0 . 0415 ( B ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure

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

B NRVMs were infected with recombinant adenoviruses encoding EGFP (AdGFP) or RGS16 24 hours prior to stimulation, then Rac1 activity was measure Data information: In (A-C) representative images from at least 3 independent experiments are shown. In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS16, P=0.0415 (B ), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4


C Cells were pretreated with 100 ng / ml pertussis toxin ( PTX , Gi / o - inhibitor ) for 24 hours or with 50 μM NSC23766 ( NSC , Tiam1 - Rac1 interaction inhibitor ) for 2 hours . Thereafter Rac1 activation was determined by effector pulldown assay Data information : In ( A - C ) representative images from at least 3 independent experiments are shown In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS1 PTX , P = 0 . 039 ( C ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure

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C Cells were pretreated with 100 ng/ml pertussis toxin (PTX, Gi/o-inhibitor) for 24 hours or with 50 μM NSC23766 (NSC, Tiam1-Rac1 interaction inhibitor) for 2 hours. Thereafter Rac1 activation was determined by effector pulldown assay Data information: In (A-C) representative images from at least 3 independent experiments are shown In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS1 PTX, P=0.039 (C), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4


D NRVMs were transduced with the 3xMEF2 - Luc reporter and were serum - starved for 20 hours . After 2 - hour of 50 μM NSC23766 ( A ) pretreatments , the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours , then MEF2 activity was determined . NSC abolished the MEF2 activation by PGE2 Data information In ( B - D ) values are mean ± s . e . m . ( D ) , n = 3 ; * , P < 0 . 05 ( Two - Way ANOVA ,

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D NRVMs were transduced with the 3xMEF2-Luc reporter and were serum-starved for 20 hours. After 2-hour of 50 μM NSC23766 (A) pretreatments, the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours, then MEF2 activity was determined. NSC abolished the MEF2 activation by PGE2 Data information In (B-D) values are mean±s.e.m. (D), n=3; *,P<0.05 (Two-Way ANOVA, RGS1


A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour . Total and phosphorylated PKD levels were analyzed by immunoblot . PKD was activated by PGE2 , but it was not affected by NSC Data information : In ( A - C ) representative images from 3 independent experiments are shown

O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC Data information: In (A-C) representative images from 3 independent experiments are shown


B After infection with Flag - HDAC5 and serum starvation , NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation . The cells were stimulated with 1 μM PGE2 for 1 hour . HDAC5 phosphorylation at Ser - 498 was detected by immunoblot . The phosphorylation of HDAC5 at this site was not prevented by NSC Data information : In ( A - C ) representative images from 3 independent experiments are shown

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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC Data information: In (A-C) representative images from 3 independent experiments are shown


C NRVMs and neonatal cardiac fibroblasts ( CFs ) were serum - starved , pretreated with vehicle or 200 nM L798106 ( EP3 receptor antagonist ) for 20 minutes , and were stimulated with 1 μM PGE2 for 10 minutes . PAK2 and PKD phosphorylation was assessed by immunoblot . Calsequestrin was used as a myocyte - marker Data information : In ( A - C ) representative images from 3 independent experiments are shown

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 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

C NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker Data information: In (A-C) representative images from 3 independent experiments are shown


D MEF2 activity was determined in cells infected with the 3xMEF2 - Luc reporter after serum starvation . Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1 - hour of 30 μM IPA - 3 pretreatment . IPA - 3 inhibited the MEF2 activation by PGE2 Data information In ( D ) values are mean ± s . e . m . , n = 3 , biological replicates , * , P < 0 . 05 ( two - way ANOVA , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure

O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O

D MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2 Data information In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5


Top : GFP - HDAC5 infected NRVMs were serum - starved for a day , pretreated for two hours with the indicated compounds ( BPKDi , 3 µM , IPA - 3 , 30 µM ) alone or in combination , then were stimulated for 4 hours . Representative images . Scale bar is 10 µm . Bottom : Statistical analysis of cellular localization of HDAC5 . GFP - HDAC5 localization ( nuclear or cytosolic ) was assessed in > 100 cells of each sample ( n = 3 , technical replicates ) . * , P < 0 , 05 ( one - way ANOVA with Bonferroni post - hoc test , vehicle vs . ET1 or vs . PGE2 , P < 0 . 0001 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0004 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0114 ; PGE2 vs . PGE2 + BPKDi + IPA - 3 , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure 6

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours A mRNA - levels of different inflammatory cytokines , as indicated . The graphs show relative mRNA - levels , fold increase compared to saline - treated controls , normalized to 18s - content Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values : IL6 , P = 0 . 0007 ( A ) ; TNFα , P < 0 . 0001 ( A )

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

MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours A mRNA-levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA-levels, fold increase compared to saline-treated controls, normalized to 18s-content Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values: IL6, P=0.0007 (A); TNFα, P<0.0001 (A)


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano - liquid chromatography tandem mass spectrometry Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values PGE2 , P = 0 . 0016 (

O 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 B-MEASURED_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 O O O O O O O O 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-MEASURED_VAR O O O O O O O

MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano-liquid chromatography tandem mass spectrometry Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values PGE2, P=0.0016 (B


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β - galactosidase - activity in MEF2 - lacZ reporter mice show MEF2 - activation ( blue cells and precipitates , respectively ) in the myocardium upon LPS - treatment . Saline - treated littermates served as control . Scale bar of histological stainings is 100 µm . Quantification of whole - heart stainings is shown in the right panel ( pixel intensity in blue channel normalized to total intensity ) Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant . In ( C ) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments . P values blue pixel intensity , P = 0 . 0083 ( C )

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

MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β-galactosidase-activity in MEF2-lacZ reporter mice show MEF2-activation (blue cells and precipitates, respectively) in the myocardium upon LPS-treatment. Saline-treated littermates served as control. Scale bar of histological stainings is 100 µm. Quantification of whole-heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity) Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant. In (C) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments. P values blue pixel intensity, P=0.0083 (C)


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD , HDAC5 and PAK2 in myocardial inflammations . Immunoblots were performed on extracts of hearts of saline - and LPS - treated mice . Representative blots are shown in the left panel , the quantification is in the right panel Data information : values are mean ± s . e . m The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values ) ; phospho - PKD - Ser - 744 / Ser - 748 , P = 0 . 0049 ( D ) ; phospho - PKD - Ser - 916 , P = 0 . 2684 ( D ) ; phospho - HDAC5 - Ser - 498 ( D ) , P = 0 . 0019 ; phospho - HDAC5 - Ser - 259 , P = 0 . 0003 ( D ) ; phospho - PAK2 - Ser - 192 / Ser - 197 , P = 0 . 1962 ( D ) ; phospho - PAK2 - Thr - 402 , P = 0 . 0446 ( D ) . The exact n and P values can be also found in the Source Data excel file for Figure

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

MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD, HDAC5 and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, the quantification is in the right panel Data information: values are mean±s.e.m The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values ); phospho-PKD-Ser-744/Ser-748, P=0.0049 (D); phospho-PKD-Ser-916, P=0.2684 (D); phospho-HDAC5-Ser-498 (D), P=0.0019; phospho-HDAC5-Ser-259, P=0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P=0.1962 (D); phospho-PAK2-Thr-402, P=0.0446 (D). The exact n and P values can be also found in the Source Data excel file for Figure 7


( a ) Diagrams of the deletion - mutation constructs of Keap1 ( left ) and the corresponding immunoprecipitation assays ( right ) . Each Flag - tagged mouse Keap1 and mutant was expressed in HEK293T cells . At 22 h after transfection , lysates were prepared and immunoprecipitated with anti - Flag antibody . The resulting immunoprecipitates were subjected to SDS - PAGE and analysed by immunoblotting with anti - Flag , anti - p62 , anti - Nrf2 and anti - actin antibodies . Data are representative of three individual experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagrams of the deletion-mutation constructs of Keap1 (left) and the corresponding immunoprecipitation assays (right). Each Flag-tagged mouse Keap1 and mutant was expressed in HEK293T cells. At 22 h after transfection, lysates were prepared and immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62, anti-Nrf2 and anti-actin antibodies. Data are representative of three individual experiments.


( b ) Diagrams of the deletion - mutation constructs of p62 ( left ) and the corresponding input ( upper right ) and pull - down assay ( lower right ) . The MBP - tagged mouse p62 deletion mutants conjugated to amylose ( AM ) resins were incubated with purified GST - tagged mouse Keap1 - DC . The pulled - down complexes with the MBP - p62 mutants were subjected to SDS - PAGE and revealed by staining with Coomassie brilliant blue . The bands corresponding to MBP - p62 and its mutants are indicated by black dots . Red arrowheads indicate the band corresponding to GST - Keap1 - DC . For details of construct 14 see Supplementary Information , Fig . S3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagrams of the deletion-mutation constructs of p62 (left) and the corresponding input (upper right) and pull-down assay (lower right). The MBP-tagged mouse p62 deletion mutants conjugated to amylose (AM) resins were incubated with purified GST-tagged mouse Keap1-DC. The pulled-down complexes with the MBP-p62 mutants were subjected to SDS-PAGE and revealed by staining with Coomassie brilliant blue. The bands corresponding to MBP-p62 and its mutants are indicated by black dots. Red arrowheads indicate the band corresponding to GST-Keap1-DC. For details of construct 14 see Supplementary Information, Fig. S3.


( d ) Immunoprecipitation assays . Flag - tagged p62 , KIR - deleted p62 ( p62 ΔKIR ) and a p62 mutant defective in oligomerization ( p62 K7AD69A ) were expressed in primary mouse hepatocytes by the adenovirus system ( left ) or in HEK293T cells by transfection ( right ) . Cell lysates were immunoprecipitated with anti - Flag antibody . The resulting immunoprecipitates were subjected to SDS - PAGE and analysed by immunoblotting with anti - p62 and anti - Keap1 antibodies . The bands corresponding to Flag - p62 , endogenous p62 , Keap1 and actin are indicated . The data shown are representative of three separate experiments .

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

(d) Immunoprecipitation assays. Flag-tagged p62, KIR-deleted p62 (p62 ΔKIR) and a p62 mutant defective in oligomerization (p62 K7AD69A) were expressed in primary mouse hepatocytes by the adenovirus system (left) or in HEK293T cells by transfection (right). Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-p62 and anti-Keap1 antibodies. The bands corresponding to Flag-p62, endogenous p62, Keap1 and actin are indicated. The data shown are representative of three separate experiments.


( e ) Interaction of endogenous p62 with Keap1 . Lysates prepared from the human hepatocellular carcinoma cell line Huh - 1 were immunoprecipitated with anti - p62 antibody or anti - Flag antibody ( negative control ) followed by immunoblotting with antibodies against p62 and Keap1 . The bands corresponding to endogenous p62 , Keap1 and IgG heavy chain ( IgG H . C . ) are indicated . The data shown are representative of three separate experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 .

(e) Interaction of endogenous p62 with Keap1. Lysates prepared from the human hepatocellular carcinoma cell line Huh-1 were immunoprecipitated with anti-p62 antibody or anti-Flag antibody (negative control) followed by immunoblotting with antibodies against p62 and Keap1. The bands corresponding to endogenous p62, Keap1 and IgG heavy chain (IgG H.C.) are indicated. The data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( a ) A representative ITC profile of the titration of Keap1 - DC with p62M7 ( residues 168 - 391 ) . The upper panel shows the raw ITC thermograms and the lower panel shows the fitted binding isotherms .

O O O O O O O O O O O O O O O O O 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) A representative ITC profile of the titration of Keap1-DC with p62M7 (residues 168-391). The upper panel shows the raw ITC thermograms and the lower panel shows the fitted binding isotherms.


( b ) Immunoprecipitation assays . Flag - tagged Keap1 was co - expressed with increasing concentrations of green fluorescent protein ( GFP ) - p62 ( lanes 3 - 6 ) in HEK293T cells . Cell lysates were immunoprecipitated with anti - Flag antibody . The resulting immunoprecipitates were subjected to SDS - PAGE and analysed by immunoblotting with anti - Flag , anti - p62 and anti - Nrf2 antibodies . The bands corresponding to Flag - Keap1 , endogenous p62 , Nrf2 and actin are indicated . Data are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation assays. Flag-tagged Keap1 was co-expressed with increasing concentrations of green fluorescent protein (GFP)-p62 (lanes 3-6) in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62 and anti-Nrf2 antibodies. The bands corresponding to Flag-Keap1, endogenous p62, Nrf2 and actin are indicated. Data are representative of three independent experiments.


( c ) The competitive p62 activity against Keap1 was measured by luciferase assay . The expression plasmids for Nrf2 , Keap1 and p62 wild - type ( w . t . ) or its mutants were transfected into Hepa1 cells along with pNqo1 - ARE reporter plasmid and pRL - TK as an internal control . At 36 h after transfection , the luciferase activity was measured in accordance with the instructions provided by the manufacturer . Assays were performed twice in triplicate . Data are means and s . d . for six determinations .

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

(c) The competitive p62 activity against Keap1 was measured by luciferase assay. The expression plasmids for Nrf2, Keap1 and p62 wild-type (w.t.) or its mutants were transfected into Hepa1 cells along with pNqo1-ARE reporter plasmid and pRL-TK as an internal control. At 36 h after transfection, the luciferase activity was measured in accordance with the instructions provided by the manufacturer. Assays were performed twice in triplicate. Data are means and s.d. for six determinations.


( d ) Immunoblot analysis . Flag - tagged p62 and its mutants defective in interacting with Keap1 were overproduced in wild - type and p62 − / − primary mouse hepatocytes by the adenovirus system . At 48 h after infection , total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the antibodies specified . The bands corresponding to Flag - p62 , endogenous p62 , Keap1 , Nrf2 , Nqo1 , actin and Lamin B are shown . Data are representative of three independent experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 .

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

(d) Immunoblot analysis. Flag-tagged p62 and its mutants defective in interacting with Keap1 were overproduced in wild-type and p62−/− primary mouse hepatocytes by the adenovirus system. At 48 h after infection, total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the antibodies specified. The bands corresponding to Flag-p62, endogenous p62, Keap1, Nrf2, Nqo1, actin and Lamin B are shown. Data are representative of three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( e ) Quantification of mRNA levels of the detoxification enzymes Nqo1 , Gstm1 and Cyp2a5 in hepatocytes overexpressing Flag - p62 and its mutants . Total RNAs were prepared from non - infected or infected hepatocytes and reverse transcribed into their respective cDNAs , which were used as templates in real - time PCR analysis . Values were normalized to the amount of each mRNA in the non - infected hepatocytes . The experiments were performed three times . Data are means ± s . d . for three experiments .

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

(e) Quantification of mRNA levels of the detoxification enzymes Nqo1, Gstm1 and Cyp2a5 in hepatocytes overexpressing Flag-p62 and its mutants. Total RNAs were prepared from non-infected or infected hepatocytes and reverse transcribed into their respective cDNAs, which were used as templates in real-time PCR analysis. Values were normalized to the amount of each mRNA in the non-infected hepatocytes. The experiments were performed three times. Data are means ± s.d. for three experiments.


( a ) Insolubilization of Keap1 in Atg7 - deficient hepatocytes . Liver homogenates from Atg7F / F : Mx1 mice on various days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble and detergent - insoluble fractions with 0 . 5 % Triton X - 100 . Each fraction was subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies . The data displayed are representative of three separate experiments .

O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O

(a) Insolubilization of Keap1 in Atg7-deficient hepatocytes. Liver homogenates from Atg7F/F:Mx1 mice on various days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. The data displayed are representative of three separate experiments.


( b ) Immunoblot analysis of Atg7 - deficient ( Atg7F / F : Mx1 ; Atg7F / F shown here as F / F ) and Atg7 p62 - deficient ( Atg7F / F : Mx1 : p62 − / − ) livers . Liver homogenates from mice of the stated genotypes at 12 days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble and detergent - insoluble fractions . Each fraction was subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies . Atg7F / F mice in which Atg7 is efficiently expressed at a level similar to that in the wild - type mice were used as control . Data shown are representative of three separate experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O 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) Immunoblot analysis of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 p62-deficient (Atg7F/F:Mx1:p62−/−) livers. Liver homogenates from mice of the stated genotypes at 12 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Atg7F/F mice in which Atg7 is efficiently expressed at a level similar to that in the wild-type mice were used as control. Data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( c ) Quantitative real - time PCR analyses of Nqo1 and Gstm1 in mouse livers . Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly ( I ) • poly ( C ) . Values were normalized to the amount of mRNA in Atg7F / F liver . Data are means ± s . d . for three experiments .

O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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

(c) Quantitative real-time PCR analyses of Nqo1 and Gstm1 in mouse livers. Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in Atg7F/F liver. Data are means ± s.d. for three experiments.


( d ) Immunofluorescence analysis of the cellular localization of p62 and Keap1 . Liver sections from mice of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) were immunostained with anti - Keap1 ( top ) and anti - p62 ( middle ) antibodies . Bottom : merged images of Keap1 ( green ) and p62 ( red ) . Each inset in the Atg7 - deficient liver panels is a magnified image of the boxed region . Scale bars , 20 μm .

O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O

(d) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (top) and anti-p62 (middle) antibodies. Bottom: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.


( a ) Immunoblotting of Atg7 - deficient ( Atg7F / F : Mx1 ; Atg7F / F shown here as F / F ) and Atg7 Nrf2 - deficient ( Atg7F / F : Mx1 : Nrf2 − / − ) livers . Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble and detergent - insoluble fractions . Total , soluble and insoluble fractions were subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies ( top section ) . Total lysates were subjected to SDS - PAGE and analysed by immunoblotting with antibodies against Nqo1 , Gstm1 and actin ( bottom left section ) . Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) , subjected to SDS - PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B ( as control ) ( bottom right section ) . Data were obtained from three independent experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblotting of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 Nrf2-deficient (Atg7F/F:Mx1:Nrf2−/−) livers. Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1, Gstm1 and actin (bottom left section). Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C), subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B (as control) (bottom right section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( b ) Immunofluorescence analysis of the cellular localization of p62 and Keap1 . Liver sections from mice of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) were immunostained with anti - Keap1 ( left ) and anti - p62 ( middle ) antibodies . Right : merged images of Keap1 ( green ) and p62 ( red ) . Each inset in the Atg7 - deficient and Atg7 Nrf2 - deficient liver panels is a magnified image of the boxed region . Scale bars , 20 μm .

O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O

(b) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (left) and anti-p62 (middle) antibodies. Right: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient and Atg7 Nrf2-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.


( c ) Quantitative real - time PCR analyses of Nqo1 , Gstm1 and Cyp2a5 in mouselivers . Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) . Values were normalized to the amount of mRNA in the Atg7F / F liver . Data are means ± s . d . for three experiments .

O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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

(c) Quantitative real-time PCR analyses of Nqo1, Gstm1 and Cyp2a5 in mouselivers. Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in the Atg7F/F liver. Data are means ± s.d. for three experiments.


( d ) Liver weight . The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly ( I ) • poly ( C ) were measured . Data are means ± s . d . for five mice from each group . Three asterisks , P 0 . 001 ( Student ' s t - test ) .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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

(d) Liver weight. The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly(I)•poly(C) were measured. Data are means ± s.d. for five mice from each group. Three asterisks, P 0.001 (Student's t-test).


( e ) Histological analysis of the mouse liver of the indicated genotypes . At 28 days after injection of poly ( I ) • poly ( C ) , the livers were processed for haematoxylin / eosin staining . Higher - magnification views are shown in the bottom panels . CV , central vein ; P , portal vein . Scale bars , 100 μm .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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

(e) Histological analysis of the mouse liver of the indicated genotypes. At 28 days after injection of poly(I)•poly(C), the livers were processed for haematoxylin/eosin staining. Higher-magnification views are shown in the bottom panels. CV, central vein; P, portal vein. Scale bars, 100 μm.


( f ) Liver function tests of the mice used in d . The serum levels of aspartate aminotransferase ( AST ) , alanine aminotransferase ( ALT ) and alkaline phosphatase ( ALP ) were measured . ( IU / L , international unit per liter ) . Data are means ± s . d . for seven mice from each group . Asterisk , P 0 . 05 ; three asterisks , P 0 . 001 .

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

(f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured. (IU/L, international unit per liter). Data are means ± s.d. for seven mice from each group. Asterisk, P 0.05; three asterisks, P 0.001.


( a ) Immunoblotting of Keap1 - deficient ( Keap1F / F : Alb ) and Keap1 Atg7 - deficient ( Keap1F / F : Atg7F / F : Alb ) livers . Liver homogenates from 8 - week - old mice of these genotypes were separated into detergent - soluble and detergent - insoluble fractions . Total , soluble and insoluble fractions were subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies ( top section ) . Total lysates were subjected to SDS - PAGE and analysed by immunoblotting with antibodies against Nqo1 and actin ( bottom section ) . Data were obtained from three independent experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblotting of Keap1-deficient (Keap1F/F:Alb) and Keap1 Atg7-deficient (Keap1F/F:Atg7F/F:Alb) livers. Liver homogenates from 8-week-old mice of these genotypes were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1 and actin (bottom section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( b ) Quantitative real - time PCR analyses of Nqo1 , Mrp4 , p62 and Ho - 1 in mouse livers . Total RNAs were prepared from livers of 9 - week - old indicated genotypes . Values were normalized to the amount of mRNA in the Atg7F / F liver ( control ) . Data are means ± s . d . for three experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative real-time PCR analyses of Nqo1, Mrp4, p62 and Ho-1 in mouse livers. Total RNAs were prepared from livers of 9-week-old indicated genotypes. Values were normalized to the amount of mRNA in the Atg7F/F liver (control). Data are means ± s.d. for three experiments.


( c ) Kaplan - Meier curves of survival of Atg7F / F : Alb and Keap1F / F : Atg7F / F : Alb mice . The survival analysis of control ( n = 33 ) , Keap1F / F : Alb ( n = 15 ) , Atg7F / F : Alb ( n = 12 ) and Keap1F / F : Atg7F / F : Alb mice ( n = 14 ) was based on a 16 - week follow - up period .

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

(c) Kaplan-Meier curves of survival of Atg7F/F:Alb and Keap1F/F:Atg7F/F:Alb mice. The survival analysis of control (n = 33), Keap1F/F:Alb (n = 15), Atg7F/F:Alb (n = 12) and Keap1F/F:Atg7F/F:Alb mice (n = 14) was based on a 16-week follow-up period.


( d ) Liver weight relative to body weight was measured for the different genotypes . Data are means ± s . d . for Atg7F / F ( control ) ( n = 24 ) , Keap1F / F : Alb ( n = 13 ) , Atg7F / F : Alb ( n = 9 ) and Keap1F / F : Atg7F / F : Alb mice ( n = 7 ) . Two asterisks , P 0 . 01 ( Student ' s t - test ) .

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

(d) Liver weight relative to body weight was measured for the different genotypes. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01 (Student's t-test).


( e ) Histological analysis of mouse liver of the indicated genotypes . The livers from 9 - week - old mice were processed for haematoxylin / eosin staining . Scale bar , 50 μm ( left bottom panel ) ; 20 μm ( other panels ) .

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

(e) Histological analysis of mouse liver of the indicated genotypes. The livers from 9-week-old mice were processed for haematoxylin/eosin staining. Scale bar, 50 μm (left bottom panel); 20 μm (other panels).


( f ) Liver function tests of the mice used in d . The serum levels of aspartate aminotransferase ( AST ) and alanine aminotransferase ( ALT ) were measured . Data are means ± s . d . for Atg7F / F ( control ) ( n = 24 ) , Keap1F / F : Alb ( n = 13 ) , Atg7F / F : Alb ( n = 9 ) and Keap1F / F : Atg7F / F : Alb mice ( n = 7 ) . Two asterisks , P 0 . 01 .

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

(f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01.


( g ) Immunohistochemical analysis of cellular localization of p62 and Keap1 . Paraffin sections of the liver from 9 - week - old mice of the indicated genotypes were immunolabelled with anti - p62 ( left ) and anti - Keap1 ( right ) antibodies . Scale bars , 20 μm .

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

(g) Immunohistochemical analysis of cellular localization of p62 and Keap1. Paraffin sections of the liver from 9-week-old mice of the indicated genotypes were immunolabelled with anti-p62 (left) and anti-Keap1 (right) antibodies. Scale bars, 20 μm.


( C - G ) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course ( right panels ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour ( 60 + ) , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52 - YFP foci ( 60 - ) . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( left panels ) . Cell cycle progression was determined by cell sorting . The mean and SEM of 3 ( wild type , rad18 ∆ , mms2 ∆ , rad51 ∆ , rad57 ∆ , rad54 ∆ ) , 4 ( pol30 - K164R ) and 5 ( rad5 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where three asterisks represent p - values < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.


( C - E ) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course ( D and E , right panel ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( C and E , left panel ) . Data information : The mean and SEM of and 3 - 12 12 ( wild type ) , 6 ( rev1 ∆ , rev3 ∆ , rad30 ∆ , rev1 ∆ rad30 ∆ , rev3 ∆ rad30 ∆ ) , and 3 ( rev1 ∆ rev3 ∆ and rev1 ∆ rev3 ∆ rad30 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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

(C-E) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel). Data information: The mean and SEM of and 3-12 12 (wild type), 6 (rev1∆, rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆ rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( C - D ) ssDNA repair efficiency of the rad5 ∆ and rev1 ∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course ( right panels ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( left panels ) . Data information : The mean and SEM of independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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

(C-D) ssDNA repair efficiency of the rad5∆ and rev1∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Data information: The mean and SEM of independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


fluorescence signal of RPA foci per cell ( E ) at times 1 or 2 ( peak ) and 7 hours from the time course shown in ( B ) . Data information : The mean and SEM of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . One , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

fluorescence signal of RPA foci per cell (E) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( F ) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1 - YFP at the ARS305 replication origin . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium . The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release . RPA enrichment was calculated as the ratio between immunoprecipitate and input values . All values were normalized to the wild type at 7 hours , taken as 1 . Data information : The mean and SEM of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . One , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively . In ( F ) all values were significant relative to the untagged controls ( not shown for clarity ) .

O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release. RPA enrichment was calculated as the ratio between immunoprecipitate and input values. All values were normalized to the wild type at 7 hours, taken as 1. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).


Effect of the lack of HR factors in spontaneous and MMS - induced mutagenesis . The frequency of spontaneous and MMS - induced forward mutagenesis at the CAN1 locus was determined in mid - log phase cell cultures before ( spontaneous mutagenesis ) and after treatment with 0 . 01 % MMS for 4 hours ( DNA damage - induced ) . Thus , the frequency of DNA damage - induced mutagenesis includes the mutants that arose spontaneously before MMS addition . The frequency of mutagenesis was determined at 0 . 01 % MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine - resistant cells . Survival ( % ) after 4 hours in MMS relative to the wild type ( taken as 100 ) is shown below the genotypes . The mean and SEM of 6 ( wild type ) , 9 ( rad54 ∆ ) and 3 ( rest ) independent fluctuation tests are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test ( one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively ) . Only the statistical analysis of the effect of rad52 ∆ , rad57 ∆ and rad51 ∆ on rad54 ∆ - mediated mutagenesis is included for clarity . All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS .

O O O O O O O O O O B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Effect of the lack of HR factors in spontaneous and MMS-induced mutagenesis. The frequency of spontaneous and MMS-induced forward mutagenesis at the CAN1 locus was determined in mid-log phase cell cultures before (spontaneous mutagenesis) and after treatment with 0.01% MMS for 4 hours (DNA damage-induced). Thus, the frequency of DNA damage-induced mutagenesis includes the mutants that arose spontaneously before MMS addition. The frequency of mutagenesis was determined at 0.01% MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine-resistant cells. Survival (%) after 4 hours in MMS relative to the wild type (taken as 100) is shown below the genotypes. The mean and SEM of 6 (wild type), 9 (rad54∆) and 3 (rest) independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test (one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively). Only the statistical analysis of the effect of rad52∆, rad57∆ and rad51∆ on rad54∆-mediated mutagenesis is included for clarity. All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS.


( A ) Effect of the rad54 ∆ and rad52 ∆ mutations in spontaneous and UV - induced uSCE . The frequency of spontaneous and UV - induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions ( SMM and SMM without histidine to calculate total and recombinant cells , respectively ) . The mean and SEM of 3 - 5 independent fluctuation tests are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . Two and three asterisks represent p - values < 0 . 01 and < 0 . 001 , respectively .

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

(A) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced uSCE. The frequency of spontaneous and UV-induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM without histidine to calculate total and recombinant cells, respectively). The mean and SEM of 3-5 independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. Two and three asterisks represent p-values <0.01 and <0.001, respectively.


( B ) Rad6 binds to DNA in the absence and presence of MMS . ChEC analysis of G1 - synchronyzed cells released in the absence or the presence of 0 . 033 % MMS for the indicated times . Data information : DNA gel electrophoresis , DNA content ( cell sorting ) and quantification of DNA digestion profiles are shown . The experiments were repeated twice with similar results .

O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(B) Rad6 binds to DNA in the absence and presence of MMS. ChEC analysis of G1-synchronyzed cells released in the absence or the presence of 0.033% MMS for the indicated times. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( C - F ) Rad6 binding to DNA depends on Rad18 , Rad52 ( C ) , Rad51 ( D ) , Rad57 ( E ) but not Rad54 ( F ) as determined by ChEC analyses of asynchronous cell cultures without or with 0 . 05 % MMS for 2 hours . Data information : DNA gel electrophoresis , DNA content ( cell sorting ) and quantification of DNA digestion profiles are shown . The experiments were repeated twice with similar results .

O O O O O O O O B-MEASURED_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 B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(C-F) Rad6 binding to DNA depends on Rad18, Rad52 (C), Rad51 (D), Rad57 (E) but not Rad54 (F) as determined by ChEC analyses of asynchronous cell cultures without or with 0.05% MMS for 2 hours. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( A - D ) Effect of rad52 ∆ , rad51 ∆ ( A ) , rad57 ∆ ( B ) and rad54 ∆ ( C ) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0 . 025 % MMS . Cell cycle progression was followed by cell sorting analysis and western blot against Clb2 . Pgk1 was used as loading control . The amounts of mono - and polyubiquitylated PCNA were normalized to the total amount of PCNA . For each time point , rad mutant values were relativized to those of the wild type , taken as 1 . The mean and SEM of 3 independent experiments are shown ( D ) . An asterisk indicates a mean significantly different than 1 , according to a One - sample t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Effect of rad52∆, rad51∆ (A), rad57∆ (B) and rad54∆ (C) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0.025% MMS. Cell cycle progression was followed by cell sorting analysis and western blot against Clb2. Pgk1 was used as loading control. The amounts of mono- and polyubiquitylated PCNA were normalized to the total amount of PCNA. For each time point, rad mutant values were relativized to those of the wild type, taken as 1. The mean and SEM of 3 independent experiments are shown (D). An asterisk indicates a mean significantly different than 1, according to a One-sample t-test.


Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC , treated with 1 % ( w / v ) digitonin and F - ATP synthase was immunoprecipitated with an anti - F - ATP synthase Ab followed by 15 % SDS - PAGE . β subunit and CyPD content were detected by Western blotting . The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel , referring to pH 7 . 4 values as 100 % . Data are an average ± s . e . of 3 independent experiments .

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

Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC, treated with 1% (w/v) digitonin and F-ATP synthase was immunoprecipitated with an anti-F-ATP synthase Ab followed by 15% SDS-PAGE. β subunit and CyPD content were detected by Western blotting. The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel, referring to pH 7.4 values as 100%. Data are an average ± s.e. of 3 independent experiments.


B , C EDTA - SMP were solubilized at the indicated pH values , extracted with 1 % ( w / v ) digitonin and subjected to BN - PAGE in order to separate dimers ( Vd ) and monomers ( Vm ) , which were identified by Coomassie blue ( panel B ) or in - gel activity staining ( panel C ) and analyzed by densitometry , which is reported in the bottom part of each panel . Values report the dimer / monomer ratio , where the ratio at pH 7 . 4 was taken as 100 % . Data are an average ± s . e . of 3 independent experiments . The p - values calculated with the Student t test are shown * p ≤ 0 . 05 * * * p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C EDTA-SMP were solubilized at the indicated pH values, extracted with 1% (w/v) digitonin and subjected to BN-PAGE in order to separate dimers (Vd) and monomers (Vm), which were identified by Coomassie blue (panel B) or in-gel activity staining (panel C) and analyzed by densitometry, which is reported in the bottom part of each panel. Values report the dimer/monomer ratio, where the ratio at pH 7.4 was taken as 100%. Data are an average ± s.e. of 3 independent experiments. The p-values calculated with the Student t test are shown * p≤0.05 *** p≤0.001.


D Sequence of peptides 95 - 113 of OSCP before ( left panel ) and after reaction with DPC ( right panel ) obtained from tryptic digests of the SDS - PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F - ATP synthase . Fragments of the series b and y identified in the LC - MS / MS analysis are indicated on the sequence of the peptides . H112 and H112CeT are indicated in red boldface . Ions y4 - y17 show a mass shift of + 72 Da in the modified peptide .

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

D Sequence of peptides 95-113 of OSCP before (left panel) and after reaction with DPC (right panel) obtained from tryptic digests of the SDS-PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F-ATP synthase. Fragments of the series b and y identified in the LC-MS/MS analysis are indicated on the sequence of the peptides. H112 and H112CeT are indicated in red boldface. Ions y4-y17 show a mass shift of +72 Da in the modified peptide.


Permeabilized HEK293T cells expressing wild type ( WT ) or edited ( H112Q or H112Y ) OSCP subunit were suspended in KSCN - based media at pH 7 . 4 ( left traces ) or pH 6 . 5 ( right traces ) and PTP opening was measured as the decrease in absorbance at 540 nm . Where indicated ( arrows ) 0 . 1 mM Ca2 + in the absence of further additions ( black traces ) or in the presence of 0 . 5 mM DPC ( red traces ) or 2 µM CsA ( blue traces ) was added . The bottom panel reports the difference between the absorbance readings before the addition of Ca2 + and at the end of the swelling phase . Data are mean ± s . e . of 5 experiments ( triplicate samples ) for Ca2 + alone ( black bars ) and Ca2 + + DPC ( red bars ) and of 3 experiments ( duplicate samples ) for Ca2 + + CsA ( blue bars ) . Solid bars , pH 7 . 4 , hatched bars , pH 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Permeabilized HEK293T cells expressing wild type (WT) or edited (H112Q or H112Y) OSCP subunit were suspended in KSCN-based media at pH 7.4 (left traces) or pH 6.5 (right traces) and PTP opening was measured as the decrease in absorbance at 540 nm. Where indicated (arrows) 0.1 mM Ca2+ in the absence of further additions (black traces) or in the presence of 0.5 mM DPC (red traces) or 2 µM CsA (blue traces) was added. The bottom panel reports the difference between the absorbance readings before the addition of Ca2+ and at the end of the swelling phase. Data are mean ± s.e. of 5 experiments (triplicate samples) for Ca2+ alone (black bars) and Ca2+ + DPC (red bars) and of 3 experiments (duplicate samples) for Ca2+ + CsA (blue bars). Solid bars, pH 7.4, hatched bars, pH 6.5


Mitochondria were prepared from cells , suspended in sucrose buffer at pH 7 . 4 ( solid bars ) or 6 . 5 ( hatched bars ) in the absence ( black bars ) or presence ( blue bars ) of 1 . 6 µM CsA , extracted with 1 % ( w / v ) digitonin and immunoprecipitated with an F - ATP synthase antibody . The immunoprecipitates were subjected to 12 % SDS - PAGE followed by Western blotting for CyPD and α subunit of F - ATP synthase . Each immunodetected band was analyzed by densitometry , the ratio between CyPD and α subunit was measured and expressed relative to the ratio obtained in absence of CsA at pH 7 . 4 , which was taken as 100 % . Data are average ± s . e . of 6 independent experiments .

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

Mitochondria were prepared from cells, suspended in sucrose buffer at pH 7.4 (solid bars) or 6.5 (hatched bars) in the absence (black bars) or presence (blue bars) of 1.6 µM CsA, extracted with 1% (w/v) digitonin and immunoprecipitated with an F-ATP synthase antibody. The immunoprecipitates were subjected to 12% SDS-PAGE followed by Western blotting for CyPD and α subunit of F-ATP synthase. Each immunodetected band was analyzed by densitometry, the ratio between CyPD and α subunit was measured and expressed relative to the ratio obtained in absence of CsA at pH 7.4, which was taken as 100%. Data are average ± s.e. of 6 independent experiments.


HEK293T cells were incubated in a balanced salt solution containing 2 - deoxyglucose at pH 7 . 4 ( solid bars ) or 6 . 5 ( hatched bars ) and anoxia induced by incubation in a BD GasPak EZ Anaerobe Gas - generating Pouch System ( BD Biosciences ) . Cell death was measured based on LDH release ( average ± s . e . from 8 independent experiments ) . In all panels the p values calculated with the Student t test are shown ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293T cells were incubated in a balanced salt solution containing 2-deoxyglucose at pH 7.4 (solid bars) or 6.5 (hatched bars) and anoxia induced by incubation in a BD GasPak EZ Anaerobe Gas-generating Pouch System (BD Biosciences). Cell death was measured based on LDH release (average ± s.e. from 8 independent experiments). In all panels the p values calculated with the Student t test are shown (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).


Representative current traces recorded at V ( pipette ) + 10 mV from mitoplasts derived from wild type ( WT ) or H112Q OSCP mutation - harboring cells .

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

Representative current traces recorded at V(pipette) +10 mV from mitoplasts derived from wild type (WT) or H112Q OSCP mutation-harboring cells.


Channel activity ( top ) and corresponding amplitude histograms ( bottom ) recorded in wild type mitochondria before ( WT pH 7 . 3 ) and after acidification to pH 6 . 5 ( WT pH 6 . 5 ) . Amplitude histograms were obtained from 120 second - long current traces for the corresponding pH values and the panels are from the same experiment .

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

Channel activity (top) and corresponding amplitude histograms (bottom) recorded in wild type mitochondria before (WT pH 7.3) and after acidification to pH 6.5 (WT pH 6.5). Amplitude histograms were obtained from 120 second-long current traces for the corresponding pH values and the panels are from the same experiment.


Recordings as in panel B from mitoplasts of cells harboring the OSCP H112Q replacement . Left panel , pH 7 . 3 ; middle panel , 440 seconds after acidification to pH 6 , 5 ; right panel , 30 seconds after the addition of 5 mM Ba2 + at pH 6 . 5 . Amplitude histograms were obtained from 100 second - long segments and the panels are from the same experiment . Note that both in panels B and C well - visible subconductance states can be detected in the amplitude histograms ( peaks between open levels o2 and o3 on left panels ) . Gaussian fitting has been obtained using the Origin Program set . In all panels c , closed ; o1 , o2 , o3 open states with discrete subconductance .

O O O O O O O O O O O O O O O O O O O O O O O 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 I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Recordings as in panel B from mitoplasts of cells harboring the OSCP H112Q replacement. Left panel, pH 7.3; middle panel, 440 seconds after acidification to pH 6,5; right panel, 30 seconds after the addition of 5 mM Ba2+ at pH 6.5. Amplitude histograms were obtained from 100 second-long segments and the panels are from the same experiment. Note that both in panels B and C well-visible subconductance states can be detected in the amplitude histograms (peaks between open levels o2 and o3 on left panels). Gaussian fitting has been obtained using the Origin Program set. In all panels c, closed; o1, o2, o3 open states with discrete subconductance.