figf1 ( 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 . ( 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 .

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

figf1(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.(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.


figf3 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(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.(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.(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.


figf4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(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.(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.


figf5 ( 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 . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(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.(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).(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.


figf6 ( 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 . ( 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 ) . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(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.(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).(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.(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.


Figure 1 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.


Figure 4fluorescence 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4fluorescence 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).


Figure 5Effect 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Effect 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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 7 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.


Figure 8 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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.


Figure 1 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1  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.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.


Figure 2Permeabilized 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 . 5Mitochondria 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 ) .

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Figure 2Permeabilized 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 6.5Mitochondria 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).


Figure 3Representative 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 . 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 .

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Figure 3Representative 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.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.


Figure 4The left panel depicts a cryo - EM based atomic model of monomeric bovine F - ATP synthase ( pdb 5ara ) ( excluding subunits e , f , g , DAPIT , 6 . 8PL and A6L ) with OSCP ( cyan ) and subunit b ( yellow ) highlighted on the structure of F - ATP synthase in the lower part of the panel . The region H112 - E115 is shown as van der Waals spheres ( inflated for better visibility ) . The backbone of the stretch T130 - K135 is shown in red van der Waals spheres . The top part of the panel shows the results of restrained molecular dynamics simulations of the isolated lateral stalk ( OSCP and subunits b , d , F6 ) displaying a large number of conformations in this region . Snapshots taken at 1 ns intervals along the last 15 ns of the trajectory are shown for the fragment 112 - HRGE - 115 for the deprotonated ( HIS ) and the protonated form ( HIS + ) . Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112 - E115 is found in a large number of MD conformations . The larger conformational variability of the deprotonated form can also be appreciated . The right panel reports a sequence comparison of OSCP across 16 out of 197 species ( HRGE is boxed in red ; Sauria sequences are in the thin black box ) . Conservation , quality of the aligment and consensus at each position in the alignment of all vertebrate sequences in RefSeq , excluding Sauria and most divergent sequences , are described in the documentation of the program JalView [ 67 ] .

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Figure 4The left panel depicts a cryo-EM based atomic model of monomeric bovine F-ATP synthase (pdb 5ara) (excluding subunits e, f, g, DAPIT, 6.8PL and A6L) with OSCP (cyan) and subunit b (yellow) highlighted on the structure of F-ATP synthase in the lower part of the panel. The region H112-E115 is shown as van der Waals spheres (inflated for better visibility). The backbone of the stretch T130-K135 is shown in red van der Waals spheres. The top part of the panel shows the results of restrained molecular dynamics simulations of the isolated lateral stalk (OSCP and subunits b, d, F6) displaying a large number of conformations in this region. Snapshots taken at 1 ns intervals along the last 15 ns of the trajectory are shown for the fragment 112-HRGE-115 for the deprotonated (HIS) and the protonated form (HIS+). Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112-E115 is found in a large number of MD conformations. The larger conformational variability of the deprotonated form can also be appreciated. The right panel reports a sequence comparison of OSCP across 16 out of 197 species (HRGE is boxed in red; Sauria sequences are in the thin black box). Conservation, quality of the aligment and consensus at each position in the alignment of all vertebrate sequences in RefSeq, excluding Sauria and most divergent sequences, are described in the documentation of the program JalView [67].


Figure 1I , J . Cumulative cell numbers of WT and Tfcp2l1 _ KO 2i - ESCs ( I ) and FBS - ESCs ( J ) . Results are from three independent clones of WT and Tfcp2l1 _ KO ESCs .

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Figure 1I, J. Cumulative cell numbers of WT and Tfcp2l1_KO 2i-ESCs (I) and FBS-ESCs (J). Results are from three independent clones of WT and Tfcp2l1_KO ESCs.


Figure 3A . Real - time fatty acid oxidation ( FAO ) profile of WT and Tfcp2l1 _ KO 2i - ESCs using the FAO stress test kit in Seahorse XF . Oxygen consumption rate ( OCR ) were measured . Error bars indicate SEM ; n = 5 biological repeats . Palm - BSA , palmitate conjugated BSA , a substrate of FAO ; Oligomycin , an inhibitor of ATP synthase ( complex V ) ; FCCP , Carbonyl cyanide - 4 ( trifluoromethoxy ) phenylhydrazone , a proton gradient uncoupler ; Rotenone and Antimycin , a complex I inhibitor and a complex III inhibitor , respectively .

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Figure 3A. Real-time fatty acid oxidation (FAO) profile of WT and Tfcp2l1_KO 2i-ESCs using the FAO stress test kit in Seahorse XF. Oxygen consumption rate (OCR) were measured. Error bars indicate SEM; n = 5 biological repeats. Palm-BSA, palmitate conjugated BSA, a substrate of FAO; Oligomycin, an inhibitor of ATP synthase (complex V); FCCP, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, a proton gradient uncoupler; Rotenone and Antimycin, a complex I inhibitor and a complex III inhibitor, respectively.


Figure 4F . Real - time FAO profile using the FAO Stress Test in Seahorse XF . n = 4 biological repeats ; error bars indicate SEM ; * * , p - value < 0 . 01 ; NS , not significant ; t - test . G . Percentage of dead cells measured by live - cell propidium iodide staining detecting spontaneous cell death in 2i - ESCs . n = 3 biological repeats ; error bars indicate SEM ; p - values were calculated using two - sample t - test . H . Cumulative cell number of WT , Tfcp2l1 _ KO , and Tfcp2l1 _ KO with Cpt1a overexpression ( OE ) 2i - ESCs . Error bars indicate SEM ; n = 3 biological repeats ; p - values were calculated using two - sample t - test .

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Figure 4F. Real-time FAO profile using the FAO Stress Test in Seahorse XF. n = 4 biological repeats; error bars indicate SEM; **, p-value <0.01; NS, not significant; t-test.G. Percentage of dead cells measured by live-cell propidium iodide staining detecting spontaneous cell death in 2i-ESCs. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test. H. Cumulative cell number of WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs. Error bars indicate SEM; n=3 biological repeats; p-values were calculated using two-sample t-test.


Figure 5C . Relative levels of metabolites in glycolysis and the TCA cycle . n = 9 biological repeats ; upper whiskers , maximum - the third quartile ; lower whiskers , the first quartile - minimum ; boxes , the third quartile - the first quartile ; the central line indicates the median ; p - values were calculated using two - sample t - test . D . Live - cell propidium iodide staining to measure cell death in WT , Tfcp2l1 _ KO , and Tfcp2l1 _ KO with Cpt1a overexpression ( OE ) 2i - ESCs after 5 mM 2 - DG treatment for 24 hours . n = 3 biological repeats ; error bars indicate SEM ; p - value were calculated using two - sample t - test . F . Relative levels of metabolites in glutamine metabolism . n = 9 biological repeats ; upper whiskers , maximum - the third quartile ; lower whiskers , the first quartile - minimum ; boxes , the third quartile - the first quartile ; the central line indicates the median ; p - values were calculated using two - sample t - test .

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Figure 5C. Relative levels of metabolites in glycolysis and the TCA cycle.n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.D. Live-cell propidium iodide staining to measure cell death in WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs after 5 mM 2-DG treatment for 24 hours. n=3 biological repeats; error bars indicate SEM; p-value were calculated using two-sample t-test.F. Relative levels of metabolites in glutamine metabolism. n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.


Figure 6D . 0 . 5 million of 2i - ESCs with indicated genotypes were seeded and treated with 200 nM INK - 128 on day0 . Cell numbers were counted on day2 , 4 , and 6 . n = 4 biological repeats ; error bars indicate SEM ; p - values were calculated using two - sample t - test . E . Immunoblotting of WT and Tfcp2l1 _ KO 2i - ESCs untreated or treated with 200 nM INK - 128 . F . Real - time PCR showing Cpt1a mRNA levels in WT and Tfcp2l1 _ KO 2i - ESCs in the presence and absence of 200 nM INK - 128 . n = 3 biological repeats ; error bars indicate SEM ; p - values were calculated using two - sample t - test .

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Figure 6D. 0.5 million of 2i-ESCs with indicated genotypes were seeded and treated with 200 nM INK-128 on day0. Cell numbers were counted on day2, 4, and 6. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.E. Immunoblotting of WT and Tfcp2l1_KO 2i-ESCs untreated or treated with 200 nM INK-128.F. Real-time PCR showing Cpt1a mRNA levels in WT and Tfcp2l1_KO 2i-ESCs in the presence and absence of 200 nM INK-128. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.


Figure 7D . Kaplan - Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK - 128 . n , number of blastocysts of each genotype . p - value was calculated using the log rank test . E . Kaplan - Meier curve of ex vivo WT blastocysts cultured in the M2 medium in the presence of 200 nM of INK - 128 with or without 50 µM of etomoxir ( Eto ) and / or 100 µM of octanoate , as indicated . p - values were calculated using the log rank test .

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Figure 7D. Kaplan-Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK-128. n, number of blastocysts of each genotype. p-value was calculated using the log rank test. E. Kaplan-Meier curve of ex vivo WT blastocysts cultured in the M2 medium in the presence of 200 nM of INK-128 with or without 50 µM of etomoxir (Eto) and/or 100 µM of octanoate, as indicated. p-values were calculated using the log rank test.


Figure 2Circular representation of DNA methylation levels for mothers ( outer circle ) and children ( inner circle ) . The height of each bar indicates the methylation change between the smoking and non ‐ smoking group ( dark hue : hypermethylation , light hue : hypomethylation ) .

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Figure 2Circular representation of DNA methylation levels for mothers (outer circle) and children (inner circle). The height of each bar indicates the methylation change between the smoking and non‐smoking group (dark hue: hypermethylation, light hue: hypomethylation).


f1 ( a ) WT and dnm1Δ cells expressing Vph1 - RFP and Idp1 - GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0 . 08 . The cells were then sampled daily and analysed by fluorescence microscopy ( × 100 objective ) during stationary phase mitophagy in lactate medium . Scale bar , 5 μm . ( b ) WT , dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods , and samples ( 10 OD600 units ) were taken at each time point . Protein extracts were prepared and equal protein amounts ( 20 μg ) were subjected to SDS - polyacrylamide gel electrophoresis and immunoblotting with anti - GFP antibody . ( c ) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b , using four independent experiments for each strain . Error bars indicate s . d . analysis of variance , P0 . 001 ; N = 4 .

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f1(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm.(b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody.(c) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b, using four independent experiments for each strain. Error bars indicate s.d. analysis of variance, P0.001; N=4.


f2 ( c ) Density plot of the reproducibility of biological replicates .

f2(c) Density plot of the reproducibility of biological replicates.


f3 ( a ) Ratios from proteins with quantifications from all samples were log2 transformed and z - score normalized . Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k - means . The proteins in each k - means cluster were tested for enrichment of GO CC ( indicated in red ) and GO BP ( indicated in blue ) terms . ( b ) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins . ( d ) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins .

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f3(a) Ratios from proteins with quantifications from all samples were log2 transformed and z-score normalized. Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k-means. The proteins in each k-means cluster were tested for enrichment of GO CC (indicated in red) and GO BP (indicated in blue) terms.(b) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins.(d) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins.


f4A comparison of the SILAC - derived abundance as a function of time , for several proteins that either show differential behaviour between WT and the mutants ( Idh2 , Idp1 and Aco2 ) and proteins that behave similarly between all the genetic backgrounds tested ( Hsp78 ) . Shown are mean protein ratios ( two replicates for mutants , three replicates for WT ) , error bars indicating peptide - based relative s . d .

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f4A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.


f5WT ( a ) , dnm1Δ ( b ) and atg1Δ cells ( c ) and atg32Δ cells ( d ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 were incubated in SL medium for 7 days , and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti - GFP antibodies as described in the Methods section . Positive control used in c and d was from dnm1Δ cells expressing Idp1 - GFP from a plasmid at 4 - day incubation ( Fig . 1b , lane 4 ) . Release of free GFP in WT cells is reproducibly variable between these reporters , and varies between 0 ( Idh2 ) and 60 % ( Aco2 ) . ( e ) Densitometric quantification of the release of free GFP ( as % of total signal ) in four independent experiments , comparing the results between WT and dnm1Δ cells . Bars denote s . d . ( n = 4 ) .

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

f5WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).


f6 ( a ) Cells expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 as well as a plasmid - borne ( pDJB12 ) , mitochondrially targeted RFP ( mtRFP ) were incubated in SL - leucine medium for 3 days and imaged daily . Aco2 and Idp1 show clear mitophagic targeting ( white arrows point at specific examples ) by day 3 , whereas Idh2 and Hsp78 show weak or no mitophagy . In addition , Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker , whereas Idp1 - GFP and Aco2 - GFP show a near - complete co - localization with mtRFP . Scale bar , 5 μm . ( b ) Quantification of the frequency of mitophagy from a ; the percentage of cells showing mitophagic profiles in the red ( magenta bars ; plasmid - borne mtRFP ) or green ( turquoise bars ; integrated C - terminal GFP chimerae ) was calculated for each GFP chimera . Idp - GFP , red N = 76 , green N = 82 ; Aco2 - GFP , red N = 79 , green N = 82 ; Idh2 - GFP , red N = 72 , green N = 72 ; Hsp78 - GFP , red N = 70 , green N = 70 .

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

f6(a) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.


f7WT and dnm1Δ cells expressing integrated Hsp78 - GFP and plasmid - borne mtRFP were grown on lactate medium for 3 days . Cells were imaged daily and the images were analysed using ImageJ as described in Methods . Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities , respectively . Diagonal concentrations of dots indicate spatial correlation between the signals . The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3 - day time point in the dnm1Δ mutant , relative to the WT cells . Scale bar , 5 μm .

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f7WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.


Figure 1 Figure 1 . DENND2B binds ITSN Purified GST - SH3 - containing proteins were incubated with lysates of HEK - 293T cells expressing Flag - DENND2B . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . Purified GST - SH3 domains of ITSN were incubated with lysates of HEK - 293T cells expressing Flag - DENND2B . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . Lysates from HEK - 293T cells expressing Flag - ITSN - s and GFP or GFP - DENND2B were incubated with protein A beads or protein A beads coupled to anti - GFP . Bound proteins were detected by Western blot . D , E HeLa cells expressing the indicated constructs . Flag - ITSN - s was visualized with anti - Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively . Scale bar : 5 µm . Boxed regions are magnified on the bottom . White arrows and red arrows point to colocalizing and non - colocalizing structures , respectively . D , E HeLa cells expressing the indicated constructs . Flag - ITSN - s was visualized with anti - Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively . Scale bar : 5 µm . Boxed regions are magnified on the bottom . White arrows and red arrows point to colocalizing and non - colocalizing structures , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 Figure 1. DENND2B binds ITSN  Purified GST-SH3-containing proteins were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.  Purified GST-SH3 domains of ITSN were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.Lysates from HEK-293T cells expressing Flag-ITSN-s and GFP or GFP-DENND2B were incubated with protein A beads or protein A beads coupled to anti-GFP. Bound proteins were detected by Western blot.D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.


Figure 2 Figure 2 . DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK - 293T cells were treated with DMSO or okadaic acid ( OA ) , lysed and analyzed by Western blot . B HEK - 293T cells expressing Flag - DENND2B were treated with DMSO or 250 nM OA , and cell lysates were incubated with GST - proteins . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry . Low abundance peptides not included . Refer to Table EV1 for a complete peptide list . E HEK - 293T cells expressing Flag - DENND2B were treated with DMSO , 250 nM OA or with increasing concentrations of PKD inhibitor CID755673 . Cell lysates were analyzed by Western blot . F HEK - 293T cells expressing Flag - DENND2B or control cells were treated with 250 nM OA or DMSO , incubated with protein G beads coupled to anti - Flag and analyzed by Western blot . G Lysates from HEK - 293T cells expressing Flag - DENND2B and PKD - dominant negative ( DN ) were incubated with protein G beads or protein G beads coupled to anti - Flag . Bound proteins were detected by Western blot . H HEK - 293T cells co - expressing Flag - DENND2B with pcDNA3 or PKD - constitutively active ( CA ) , were incubated with protein G beads or protein G beads coupled to anti - Flag and bound proteins were detected by Western blot . I Quantification of H where PKD - CA treatment is normalized to pcDNA3 control . Mean + / - SD . Welch ' s t - test * p = 0 . 016 , n = 6 for both treatment groups 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 Figure 2. DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK-293T cells were treated with DMSO or okadaic acid (OA), lysed and analyzed by Western blot. B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST-proteins. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry. Low abundance peptides not included. Refer to Table EV1 for a complete peptide list.E HEK-293T cells expressing Flag-DENND2B were treated with DMSO, 250 nM OA or with increasing concentrations of PKD inhibitor CID755673. Cell lysates were analyzed by Western blot.F HEK-293T cells expressing Flag-DENND2B or control cells were treated with 250 nM OA or DMSO, incubated with protein G beads coupled to anti-Flag and analyzed by Western blot.G Lysates from HEK-293T cells expressing Flag-DENND2B and PKD-dominant negative (DN) were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.H HEK-293T cells co-expressing Flag-DENND2B with pcDNA3 or PKD-constitutively active (CA), were incubated with protein G beads or protein G beads coupled to anti-Flag and bound proteins were detected by Western blot.I Quantification of H where PKD-CA treatment is normalized to pcDNA3 control. Mean +/- SD. Welch's t-test *p=0.016, n=6 for both treatment groups from 3 independent experiments.


Figure 3 Figure 3 . 14 - 3 - 3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST - 14 - 3 - 3 WT or K50E were incubated with lysates of HEK - 293T cells expressing GFP - DENND2B WT or S30A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . B HEK - 293T cells expressing Flag - DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST - 14 - 3 - 3 . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . C Quantification of B . where bound DENND2B is normalized to the SM . Mean + / - SD . Welch ' s t - test * * p = 0 . 006 n = 8 for both treatment groups from 5 independent experimentsD HEK - 293T cells expressing Flag - DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST - 14 - 3 - 3 . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . E Quantification of D . where bound DENND2B is normalized to the SM . Mean + / - SD . Welch ' s t - test * p = 0 . 040 . n = 5 for both treatment groups from 3 independent experimentsF GST - SH3A was incubated with lysates of HEK - 293T cells expressing GFP - DENND2B WT or S30A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . G Lysates of HEK - 293T cells expressing Flag - DENND2B were pre - incubated with increasing concentrations of purified 14 - 3 - 3 WT and subsequently incubated with GST - SH3A ( top panel ) or GST - Grb2 ( bottom panel ) . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , 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 O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.C Quantification of B. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test **p=0.006 n=8 for both treatment groups from 5 independent experimentsD HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.E Quantification of D. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test *p=0.040. n=5 for both treatment groups from 3 independent experimentsF GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.


Figure 4 Figure 4 . DENND2B promotes EGFR recycling A , B HeLa cells expressing the indicated constructs . Boxed regions are magnified on the bottom . Scale bars : 5 µm in top panel and 1 µm bottom panel . White arrows and arrowheads point to colocalization on vesicles and plasma membrane , respectively . Figure 4 . DENND2B promotes EGFR recycling A , B HeLa cells expressing the indicated constructs . Boxed regions are magnified on the bottom . Scale bars : 5 µm in top panel and 1 µm bottom panel . White arrows and arrowheads point to colocalization on vesicles and plasma membrane , respectively . C Transduced MCF10A cells were quantified by real - time PCR . Mean + / - SD , ANOVA with Dunnett post - hoc test * * * p = 0 . 000 for both comparisons , n = 3 from 3 independent experiments . D Transduced HEK - 293T cells expressing Flag - DENND2B were analyzed by Western blot . E Transduced MCF10A cells , were biotinylated and cell lysates were incubated with streptavidin beads . Bound proteins were detected by Western blot . F Quantification of E . where surface EGFR is normalized to total EGFR . Mean + / - SD , Anova with Dunnett post - hoc test * p = 0 . 013 and * * * p = 0 . 000 . n = 6 for all treatment groups from 4 independent experimentsG Quantification of E where total EGFR is normalized to Hsc70 . Mean + / - SD n = 3 for all treatment groups from 3 independent experimentsI MCF10A cells expressing GFP - DENND2B were labelled for surface EGFR on ice , incubated at 16 ° C to allow internalization , acid washed to remove remaining labelled - EGFR and processed for immunofluorescence to visualize the internal pool of EGFR ( post - endocytosis , top panel ) or incubated at 37 ° C to allow recycling , acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR ( post - recycling , bottom panel ) . J Quantification of I top panel , internal EGFR post - endocytosis . Mean fluorescence per cell + / - SD , n = 13 for GFP - DENND2B and n = 21 for control cells pooled from 2 independent experiments . K Quantification of I bottom panel , internal EGFR post - recycling . Mean fluorescence per cell + / - SD , n = 10 for GFP - DENND2B and n = 26 for control cells pooled from 2 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 O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 Figure 4. DENND2B promotes EGFR recycling A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.  Figure 4. DENND2B promotes EGFR recycling A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.C Transduced MCF10A cells were quantified by real-time PCR. Mean +/- SD, ANOVA with Dunnett post-hoc test ***p=0.000 for both comparisons, n=3 from 3 independent experiments.D Transduced HEK-293T cells expressing Flag-DENND2B were analyzed by Western blot.E Transduced MCF10A cells, were biotinylated and cell lysates were incubated with streptavidin beads. Bound proteins were detected by Western blot.F Quantification of E. where surface EGFR is normalized to total EGFR. Mean +/- SD, Anova with Dunnett post-hoc test *p=0.013 and ***p=0.000. n=6 for all treatment groups from 4 independent experimentsG Quantification of E where total EGFR is normalized to Hsc70. Mean +/- SD n=3 for all treatment groups from 3 independent experimentsI MCF10A cells expressing GFP-DENND2B were labelled for surface EGFR on ice, incubated at 16°C to allow internalization, acid washed to remove remaining labelled-EGFR and processed for immunofluorescence to visualize the internal pool of EGFR (post-endocytosis, top panel) or incubated at 37°C to allow recycling, acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR (post-recycling, bottom panel).J Quantification of I top panel, internal EGFR post-endocytosis. Mean fluorescence per cell +/- SD, n=13 for GFP-DENND2B and n=21 for control cells pooled from 2 independent experiments.K Quantification of I bottom panel, internal EGFR post-recycling. Mean fluorescence per cell +/- SD, n=10 for GFP-DENND2B and n=26 for control cells pooled from 2 independent experiments.