( B ) Immunoprecipitation ( IP ) of endogenous BRCA2 from benzonase - treated HEK293T cell lysates left untreated or treated with IR ( 6Gy ) and harvested 4h post - IR , as indicated . Mouse IgG was used as negative control . Immunoblot of DDX5 and BRCA2 . StainFree image of the gels before transfer were used as loading control ( cropped image is shown ) . Asterisk ( * ) indicate a non - specific band detected by anti - DDX5 antibody .

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

(B) Immunoprecipitation (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse IgG was used as negative control. Immunoblot of DDX5 and BRCA2. StainFree image of the gels before transfer were used as loading control (cropped image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5 antibody.


( C ) Left : Representative images of in situ proximity ligation assay ( PLA ) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated ( - ) or irradiated ( 4h post - IR ; 6 Gy ) . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from untreated siC cells are shown . Scale bar indicates 10 µm . Right : Quantification of the number of PLA spots per nucleus . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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

(C) Left: Representative images of in situ proximity ligation assay (PLA) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the number of PLA spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( D ) Diagram showing the BRCA2 N - terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N - terminal truncations ( BRCA2T1 , BRCA2LT2 , BRCA2LT3 ) or the 2xMBP tag . DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP , respectively . StainFree images of the gels before transfer were used as loading

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

(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control


( E ) Left : GST pulldown assay using purified BRCA2T1 and DDX5 ; MBP antibody was used for the detection of both proteins . UB : unbound ; E : eluate . Right : SDS - PAGE showing 300 ng of purified MBP - DDX5 - GST and of 2xMBP - tagged BRCA2T1 used in the pulldown assay .

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

(E) Left: GST pulldown assay using purified BRCA2T1 and DDX5; MBP antibody was used for the detection of both proteins. UB: unbound; E: eluate. Right: SDS-PAGE showing 300 ng of purified MBP-DDX5-GST and of 2xMBP-tagged BRCA2T1 used in the pulldown assay.


( A ) Left : Representative images of S9 . 6 immunofluorescence of U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . The merged images show the signal of S9 . 6 , nucleolin ( nucleoli ) antibodies and DAPI staining . Scale bar indicates 25 µm . Right : Quantification of S9 . 6 average nuclear intensity of U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . The red line in the plot indicates the median and each symbol represents the value of a single cell . The statistical significance of the difference was calculated with Mann - Whitney U - test and the p - values show the significant difference . The data represent at least 235 cells per condition from three independent experiments .

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

(A) Left: Representative images of S9.6 immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference. The data represent at least 235 cells per condition from three independent experiments.


( B ) Top : Representative images of in situ PLA performed with anti - DDX5 and S9 . 6 antibodies in EdU - labelled U2OS cells . Where indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Bottom : Quantification of PLA spots per nucleus in each condition as indicated . At least 300 cells per condition were counted from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test , the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Representative images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of PLA spots per nucleus in each condition as indicated. At least 300 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Representative screenshot of a specific genomic region showing DRIPc - seq profiles at Watson ( W ) and Crick ( C ) strands in K562 cells depleted of DDX5 ( siDDX5 ) or control cells ( siC ) from two 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

(C) Representative screenshot of a specific genomic region showing DRIPc-seq profiles at Watson (W) and Crick (C) strands in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) from two independent experiments.


( D ) DNA - RNA hybrid distribution along protein - coding genes containing DRIPc - seq peaks in both conditions ( siC and siDDX5 ) and replicates . Gene metaplots represent the mean of antisense or sense mean DRIPc - seq signal from two independent experiments in K562 cells depleted of DDX5 ( siDDX5 ) or control cells ( siC ) as indicated .

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

(D) DNA-RNA hybrid distribution along protein-coding genes containing DRIPc-seq peaks in both conditions (siC and siDDX5) and replicates. Gene metaplots represent the mean of antisense or sense mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) as indicated.


( A ) Left : Representative images of in situ PLA between S9 . 6 and γH2AX antibodies in U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from non - irradiated siC cells are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Right : Quantification of PLA spots per nucleus in each condition as indicated . At least 500 cells per condition were counted from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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

(A) Left: Representative images of in situ PLA between S9.6 and γH2AX antibodies in U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from non-irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Right: Quantification of PLA spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( B ) DRIP - qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . The experiment was performed in both untreated cells ( - OHT ) and after tamoxifen addition ( + OHT ) . The data represent the mean ± SEM from at least four independent experiments . The statistical significance of the difference was calculated with unpaired one - tail t - test and the p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DRIP-qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The experiment was performed in both untreated cells (-OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant difference.


( A ) Top : Scheme showing the experimental set up for laser irradiation in DDX5 - GFP transfected U2OS cells depleted of BRCA2 ( siBRCA2 ) or control cells ( siC ) . Middle : Live cell imaging of the recruitment of GFP - 53BP1 or DDX5 - GFP to DNA damage tracks at different time points as indicated . Exposure and processing were adjusted to best demonstrate stripes and anti - stripes . Scale bar indicates 10 µm . Bottom left : Western blot showing the siRNA mediated knock - down of BRCA2 from U2OS cells transfected with DDX5 - GFP . Bottom right : Quantification of the percentage of transfected cells that exhibit DDX5 - GFP " anti - stripe " pattern ( reduced GFP signal at DNA damage tracks compared to the signal in the nucleus ) at the times indicated in cells depleted of BRCA2 ( siBRCA2 ) or treated with control siRNA ( siC ) . The data represent the mean ± SEM from three independent experiments .

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

(A) Top: Scheme showing the experimental set up for laser irradiation in DDX5-GFP transfected U2OS cells depleted of BRCA2 (siBRCA2) or control cells (siC). Middle: Live cell imaging of the recruitment of GFP-53BP1 or DDX5-GFP to DNA damage tracks at different time points as indicated. Exposure and processing were adjusted to best demonstrate stripes and anti-stripes. Scale bar indicates 10 µm. Bottom left: Western blot showing the siRNA mediated knock-down of BRCA2 from U2OS cells transfected with DDX5-GFP. Bottom right: Quantification of the percentage of transfected cells that exhibit DDX5-GFP "anti-stripe" pattern (reduced GFP signal at DNA damage tracks compared to the signal in the nucleus) at the times indicated in cells depleted of BRCA2 (siBRCA2) or treated with control siRNA (siC). The data represent the mean ± SEM from three independent experiments.


( B ) DDX5 ChIP - qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells ( - OHT ) or after tamoxifen addition ( + OHT ) . The data represent the mean ± SEM from three independent experiments . The green line represents the background levels of DDX5 signal . The statistical significance of the difference was calculated with unpaired one - tail t - test and the p - values show the significant differences between untreated cells ( - OHT ) and after tamoxifen addition ( + OHT ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DDX5 ChIP-qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells (-OHT) or after tamoxifen addition (+OHT). The data represent the mean ± SEM from three independent experiments. The green line represents the background levels of DDX5 signal. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant differences between untreated cells (-OHT) and after tamoxifen addition (+OHT).


( C ) Top : Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) and either untreated cells ( - OHT ) or after tamoxifen addition ( + OHT ) , as indicated . Scale bar indicates 10 µm . Bottom : Quantification of the number of γH2AX foci per nucleus ( left ) and DDX5 nuclear intensity ( right ) . The data represent at least 800 cells per condition from three independent experiments . The red line in the plot indicates the median and each symbol represents the value of a single cell . The statistical significance of the difference was calculated with Mann - Whitney U - test and the p - values show the significant difference .

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

(C) Top: Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) and either untreated cells (-OHT) or after tamoxifen addition (+OHT), as indicated. Scale bar indicates 10 µm. Bottom: Quantification of the number of γH2AX foci per nucleus (left) and DDX5 nuclear intensity (right). The data represent at least 800 cells per condition from three independent experiments. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference.


( A ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 2 nM purified EGFP - MBP - BRCA2 ( top gel left ) or 10 nM purified BRCA2LT3 ( top gel right ) or 50 nM purified BRCA2T1 ( bottom gel ) . Bottom : Quantification of the unwinding experiments showing the percentage of free RNA relative to the R - loop substrate ( unwound product ) as a function of DDX5 concentration alone ( black ) or in presence of BRCA2 ( red ) or BRCA2LT3 ( blue ) or BRCA2T1 ( pink ) . The data represent the mean ± SD of at least three independent experiments . Right : SDS - PAGE showing 500 ng of purified 2XMBP - BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay .

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

(A) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 2 nM purified EGFP-MBP-BRCA2 (top gel left) or 10 nM purified BRCA2LT3 (top gel right) or 50 nM purified BRCA2T1 (bottom gel). Bottom: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2 (red) or BRCA2LT3 (blue) or BRCA2T1 (pink). The data represent the mean ± SD of at least three independent experiments. Right: SDS-PAGE showing 500 ng of purified 2XMBP-BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay.


( B ) Left : Thin layer chromatography ( TLC ) plate showing a representative ATP hydrolysis assay in which purified MBP - DDX5 - GST ( 50 - 200 nM ) was incubated with R - loop synthetic substrate and [ γ32P ] ATP in presence or absence of or 6 nM purified BRCA2LT3 . Right : Quantification of the ATP hydrolysed in each condition . No protein control was used as background . The data represent the mean ± SD from three independent experiments .

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

(B) Left: Thin layer chromatography (TLC) plate showing a representative ATP hydrolysis assay in which purified MBP-DDX5-GST (50-200 nM) was incubated with R-loop synthetic substrate and [γ32P] ATP in presence or absence of or 6 nM purified BRCA2LT3. Right: Quantification of the ATP hydrolysed in each condition. No protein control was used as background. The data represent the mean ± SD from three independent experiments.


( A ) Left : GFP pull - down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 ( WT ) or the variant T207A ( T207A ) . DLD1 BRCA2 + / + ( + / + ) cell extracts were used as control for the GFP - trap . DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2 , respectively . StainFree images of the gels before transfer was used as loading control ( cropped image is shown ) . Right : Quantification of the GFP - trap pull - down experiments calculated as the co - immunoprecipitated DDX5 with either BRCA2 WT or BRCA2 - T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP - BRCA2 . Results are presented as the fold change compared to the BRCA2 WT clone . The data represents the mean ± SD of three independent experiments . Statistical significance of the difference was calculated with unpaired t test and the p - values show the significant difference .

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

(A) Left: GFP pull-down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 (WT) or the variant T207A (T207A). DLD1 BRCA2+/+ (+/+) cell extracts were used as control for the GFP-trap. DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2, respectively. StainFree images of the gels before transfer was used as loading control (cropped image is shown). Right: Quantification of the GFP-trap pull-down experiments calculated as the co-immunoprecipitated DDX5 with either BRCA2 WT or BRCA2-T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP-BRCA2. Results are presented as the fold change compared to the BRCA2 WT clone. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.


( B ) Left : Representative images of S9 . 6 immunofluorescence of DLD1 cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . The merged images show the signal of S9 . 6 , nucleolin ( nucleoli ) antibodies and DAPI staining . Scale bar indicates 25 µm . Right : Quantification of the average nuclear intensity of S9 . 6 antibody in DLD1 BRCA2 - WT ( WT ) or BRCA2 - T207A ( T207A ) cells transfected with either a plasmid expressing GFP alone ( - RH ) or GFP - RNase H1 ( + RH ) , as indicated . The red line in the plot indicates the median and each symbol represents the value of a single cell . Data correspond to at least 170 cells per condition from two independent experiments . The statistical significance of the difference was calculated with Mann - Whitney U - test ; p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative images of S9.6 immunofluorescence of DLD1 cells bearing BRCA2 (WT) or BRCA2-T207A (T207A). The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of the average nuclear intensity of S9.6 antibody in DLD1 BRCA2-WT (WT) or BRCA2-T207A (T207A) cells transfected with either a plasmid expressing GFP alone (-RH) or GFP-RNase H1 (+RH), as indicated. The red line in the plot indicates the median and each symbol represents the value of a single cell. Data correspond to at least 170 cells per condition from two independent experiments. The statistical significance of the difference was calculated with Mann-Whitney U-test; p-values show the significant difference.


( C ) Relative DRIP - qPCR signal values ( respect to the siC level at each locus ) at the MALAT1 , RRPH1 and HIST1H2BG loci in DLD1 BRCA2 - / - cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) and treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . The data represent the mean ± SEM from seven independent experiments . The statistical significance of the difference was calculated with paired t - Student test ; the p - values show the significant difference .

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

(C) Relative DRIP-qPCR signal values (respect to the siC level at each locus) at the MALAT1, RRPH1 and HIST1H2BG loci in DLD1 BRCA2-/- cells bearing BRCA2 (WT) or BRCA2-T207A (T207A) and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The data represent the mean ± SEM from seven independent experiments. The statistical significance of the difference was calculated with paired t-Student test; the p-values show the significant difference.


( D ) Top : Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) , as indicated . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 750 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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

(D) Top: Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 (WT) or BRCA2-T207A (T207A), as indicated. Cells were fixed directly or 4h post-irradiation (6Gy). Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 750 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( A ) Top : Representative images of In situ PLA performed between DDX5 and S9 . 6 ( DNA - RNA hybrids ) antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) or DLD1 stable clones expressing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 500 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot . ( B ) Top : Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 600 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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

(A) Top: Representative images of In situ PLA performed between DDX5 and S9.6 (DNA-RNA hybrids) antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) or DLD1 stable clones expressing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 500 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. (B) Top: Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Left : Representative images of the recruitment of transient transfected GFP - MBP - BRCA2 ( WT and T207A ) to DNA damage tracks at 5 min post - irradiation in U2OS cells . γH2AX is used as a marker of DNA damage . Scale bar indicates 10 µm . Right : Quantification of the intensity of GFP signal at the laser tracks . At least 130 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the two samples we applied an unpaired t - test . The red line in the plot indicates the median and each symbol represents a single cell intensity value .

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

(C) Left: Representative images of the recruitment of transient transfected GFP-MBP-BRCA2 (WT and T207A) to DNA damage tracks at 5 min post-irradiation in U2OS cells. γH2AX is used as a marker of DNA damage. Scale bar indicates 10 µm. Right: Quantification of the intensity of GFP signal at the laser tracks. At least 130 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the two samples we applied an unpaired t-test. The red line in the plot indicates the median and each symbol represents a single cell intensity value.


( D ) Top : Representative images of in situ PLA performed between S9 . 6 ( DNA - RNA hybrids ) and anti - γH2AX antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Bottom : Quantification of the number of PLA spots per nucleus . At least 600 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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

(D) Top: Representative images of in situ PLA performed between S9.6 (DNA-RNA hybrids) and anti-γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). When indicated, cells were transfected/treated with RNase H1 (RH) or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( E ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 50 nM purified BRCA2T1 - T207A ( bottom right ) . Bottom left : Quantification of the unwinding experiments showing the percentage of free RNA relative to the R - loop substrate ( unwound product ) as a function of DDX5 concentration alone ( black ) or in presence of BRCA2T1 - T207A ( green ) . The data represent the mean ± SD of at least three independent experiments . Bottom right : SDS - PAGE showing 650 ng of purified 2XMBP - BRCA2T1 - T207A used in the unwinding assay .

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

(E) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 50 nM purified BRCA2T1-T207A (bottom right). Bottom left: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2T1-T207A (green). The data represent the mean ± SD of at least three independent experiments. Bottom right: SDS-PAGE showing 650 ng of purified 2XMBP-BRCA2T1-T207A used in the unwinding assay.


( A ) Top : Representative immunofluorescence images of cells stained for RAD51 ( green ) in U2OS cells depleted of DDX5 ( siDDX5 ) and in control cells ( siC ) in non - treated ( - ) or different time points after exposure to IR ( 6Gy ) , as indicated . Scale bar indicates 10 µm . Bottom : Graph showing the average number of RAD51 repair foci in both cell lines . The data represent the mean ± SEM of three independent experiments .

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

(A) Top: Representative immunofluorescence images of cells stained for RAD51 (green) in U2OS cells depleted of DDX5 (siDDX5) and in control cells (siC) in non-treated (-) or different time points after exposure to IR (6Gy), as indicated. Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in both cell lines. The data represent the mean ± SEM of three independent experiments.


( B ) Top : Representative immunofluorescence images of DLD1 BRCA2 deficient cells ( BRCA2 - / - ) or BRCA2 - / - cells expressing BRCA2 WT ( WT ) or BRCA2 - T207A ( T207A ) in non - treated or at different time points post - IR ( 6 Gy ) , as indicated ; hybridized with anti - RAD51 antibody ( green ) . Scale bar indicates 10 µm . Bottom : Graph showing the average number of RAD51 repair foci in the three cell lines . The data represent the mean ± SEM of three independent experiments .

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

(B) Top: Representative immunofluorescence images of DLD1 BRCA2 deficient cells (BRCA2-/-) or BRCA2-/- cells expressing BRCA2 WT (WT) or BRCA2-T207A (T207A) in non-treated or at different time points post-IR (6 Gy), as indicated; hybridized with anti-RAD51 antibody (green). Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in the three cell lines. The data represent the mean ± SEM of three independent experiments.


( C ) Left : Representative immunofluorescence images of DLD1 BRCA2 WT or BRCA2 - T207A cells 4h post - irradiation ( 6Gy ) hybridized with anti - γH2AX and anti - RPA antibodies . Right : Quantification of the number of γH2AX and RPA foci per nucleus , as indicated . The data represent at least 400 cells per condition from two independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single focus .

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

(C) Left: Representative immunofluorescence images of DLD1 BRCA2 WT or BRCA2-T207A cells 4h post-irradiation (6Gy) hybridized with anti-γH2AX and anti-RPA antibodies. Right: Quantification of the number of γH2AX and RPA foci per nucleus, as indicated. The data represent at least 400 cells per condition from two independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single focus.


( D ) Left : Representative immunofluorescence images of DLD1 BRCA2 - T207A cells 2h post - irradiation ( 6Gy ) , as indicated , hybridized with anti - γH2AX and anti - RAD51 antibodies . ( RH ) RNase H . Right : Quantification of the number of γH2AX foci ( left ) or RAD51 foci ( right ) per nucleus . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . The data shown is from at least 400 cells per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single focus .

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

(D) Left: Representative immunofluorescence images of DLD1 BRCA2-T207A cells 2h post-irradiation (6Gy), as indicated, hybridized with anti-γH2AX and anti-RAD51 antibodies. (RH) RNase H. Right: Quantification of the number of γH2AX foci (left) or RAD51 foci (right) per nucleus. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. The data shown is from at least 400 cells per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single focus.


( E ) Top : Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2 - T207A ( T207A ) in chromatin fraction . When indicated , cells were irradiated ( 2h treatment ) and / or transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . α - tubulin and Histone H3 signals are shown as markers of cytoplasmic and chromatin fraction respectively . Bottom : quantification of RAD51 levels detected by Western blot with a RAD51 specific antibody in chromatin fraction relative to RAD51 levels in WCE . Results are presented as the fold change compared to the untreated sample . The data represents the mean ± SD of three independent experiments . Statistical significance of the difference was calculated with unpaired t test and the p - values show the significant difference .

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

(E) Top: Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2-T207A (T207A) in chromatin fraction. When indicated, cells were irradiated (2h treatment) and/or transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. α-tubulin and Histone H3 signals are shown as markers of cytoplasmic and chromatin fraction respectively. Bottom: quantification of RAD51 levels detected by Western blot with a RAD51 specific antibody in chromatin fraction relative to RAD51 levels in WCE. Results are presented as the fold change compared to the untreated sample. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.


TP53INP1 - KO ( − / − ) and WT ( + / + ) male mice were subjected to a high - fat diet ( HFD , 60 % fat ) or a control diet ( CTRL ) for 16 weeks . Mice drank tap water or tap water supplemented with NAC ( 10 mg / ml or 1 % ) . Curves show mice body weight recorded every week . CTRL : P ( − / − versus + / + ; t = 8w ) = 0 . 047 ; P ( − / − versus + / + ; t = 9w ) = 0 . 023 . HFD : P ( − / − versus + / + ; t = 7w ) = 0 . 039 ; P ( − / − versus + / + ; t = 8w ) = 0 . 029 ; P ( − / − versus + / + ; t = 9w ) = 0 . 021 ; P ( − / − versus + / + ; t = 10w ) = 0 . 014 ; P ( − / − versus + / + ; t = 11w ) = 0 . 0046 ; P ( − / − versus + / + ; t = 12w ) = 0 . 0028 ; P ( − / − versus + / + ; t = 13w ) = 0 . 0025 ; P ( − / − versus + / + ; t = 14w ) = 0 . 00051 ; P ( − / − versus + / + ; t = 15w ) = 0 . 00027 ; P ( − / − versus + / + ; t = 16w ) = 0 . 00013 .

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

TP53INP1-KO (−/−) and WT (+/+) male mice were subjected to a high-fat diet (HFD, 60% fat) or a control diet (CTRL) for 16 weeks. Mice drank tap water or tap water supplemented with NAC (10 mg/ml or 1%).Curves show mice body weight recorded every week. CTRL: P (−/− versus +/+; t = 8w) = 0.047; P (−/− versus +/+; t = 9w) = 0.023. HFD: P (−/− versus +/+; t = 7w) = 0.039; P (−/− versus +/+; t = 8w) = 0.029; P (−/− versus +/+; t = 9w) = 0.021; P (−/− versus +/+; t = 10w) = 0.014; P (−/− versus +/+; t = 11w) = 0.0046; P (−/− versus +/+; t = 12w) = 0.0028; P (−/− versus +/+; t = 13w) = 0.0025; P (−/− versus +/+; t = 14w) = 0.00051; P (−/− versus +/+; t = 15w) = 0.00027; P (−/− versus +/+; t = 16w) = 0.00013.


At the end of protocol , mice were sacrificed ; liver and epididymal and renal fat masses were taken and weighed . Histograms show organ weight . Liver : P ( − / − versus + / + ; HFD ) = 0 . 014 ; P ( HFD versus CTRL ; + / + ) = 0 . 00063 ; P ( HFD versus CTRL ; − / − ) = 0 . 0010 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 034 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 027 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 014 . Epididymal fat mass : P ( − / − versus + / + ; HFD ) = 0 . 011 ; P ( HFD versus CTRL ; + / + ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ) = 0 . 000017 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 019 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 0054 ; P ( NAC versus no NAC ; + / + CTRL ) = 0 . 037 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0025 . Renal fat mass : P ( − / − versus + / + ; HFD ) = 0 . 0041 ; P ( HFD versus CTRL ; + / + ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ) = 0 . 000013 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 019 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 0078 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0047 .

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

At the end of protocol, mice were sacrificed; liver and epididymal and renal fat masses were taken and weighed. Histograms show organ weight. Liver: P (−/− versus +/+; HFD) = 0.014; P (HFD versus CTRL; +/+) = 0.00063; P (HFD versus CTRL; −/−) = 0.0010; P (HFD versus CTRL; +/+ NAC) = 0.034; P (HFD versus CTRL; −/− NAC) = 0.027; P (NAC versus no NAC; −/− HFD) = 0.014. Epididymal fat mass: P (−/− versus +/+; HFD) = 0.011; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000017; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0054; P (NAC versus no NAC; +/+ CTRL) = 0.037; P (NAC versus no NAC; −/− HFD) = 0.0025. Renal fat mass: P (−/− versus +/+; HFD) = 0.0041; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000013; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0078; P (NAC versus no NAC; −/− HFD) = 0.0047.


A , B Histograms show bloodglucose ( A ) or plasmainsulin ( B ) levels of 6 - h - fasted mice at the beginning ( Week 0 ) and / or at the end of the protocol ( Week 16 ) . Fasting bloodglucose week 16 : P ( − / − versus + / + ; HFD ) = 0 . 0052 ; P ( CTRL versus HFD ; − / − ) = 0 . 000081 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0019 ; P ( w16 versus w0 ; + / + CTRL ) = 0 . 012 ; P ( w16 versus w0 ; − / − CTRL ) = 0 . 050 ; P ( w16 versus w0 ; − / − HFD ) = 0 . 0023 . Fasting plasmainsulin : P ( − / − versus + / + ; HFD ) = 0 . 043 ; P ( CTRL versus HFD ; + / + ) = 0 . 028 ; P ( CTRL versus HFD ; − / − ) = 0 . 013 ; P ( CTRL versus HFD ; + / + NAC ) = 0 . 011 ; P ( CTRL versus HFD ; − / − NAC ) = 0 . 0015 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 038 .

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

A, B Histograms show bloodglucose (A) or plasmainsulin (B) levels of 6-h-fasted mice at the beginning (Week 0) and/or at the end of the protocol (Week 16). Fasting bloodglucose week 16: P (−/− versus +/+; HFD) = 0.0052; P (CTRL versus HFD; −/−) = 0.000081; P (NAC versus no NAC; −/− HFD) = 0.0019; P (w16 versus w0; +/+ CTRL) = 0.012; P (w16 versus w0; −/− CTRL) = 0.050; P (w16 versus w0; −/− HFD) = 0.0023. Fasting plasmainsulin: P (−/− versus +/+; HFD) = 0.043; P (CTRL versus HFD; +/+) = 0.028; P (CTRL versus HFD; −/−) = 0.013; P (CTRL versus HFD; +/+ NAC) = 0.011; P (CTRL versus HFD; −/− NAC) = 0.0015; P (NAC versus no NAC; −/− HFD) = 0.038.


C Glucose tolerance test ( GTT ) was performed on 6 - h - fasted mice during 120 min after injection of 1 g glucose / kg of body weight . Curves on the left show blood glucose level monitored after injection of glucose . Histograms on the right show area under curve ( AUC ) . Fasting blood glucose : P ( − / − versus + / + ; CTRL ; t = 0 min ) = 0 . 045 ; P ( − / − versus + / + ; CTRL ; t = 15 min ) = 0 . 013 ; P ( − / − versus + / + ; CTRL ; t = 30 min ) = 0 . 016 ; P ( − / − versus + / + ; CTRL ; t = 60 min ) = 0 . 030 ; P ( − / − versus + / + ; CTRL ; t = 90 min ) = 0 . 017 ; P ( − / − versus + / + ; HFD ; t = 60 min ) = 0 . 041 ; P ( − / − versus + / + ; HFD ; t = 90 min ) = 0 . 043 ; P ( − / − versus + / + ; HFD ; t = 120 min ) = 0 . 034 ; P ( HFD versus CTRL ; + / + ; t = 15 min ) = 0 . 0076 ; P ( HFD versus CTRL ; + / + ; t = 30 min ) = 0 . 0067 ; P ( HFD versus CTRL ; + / + ; t = 60 min ) = 0 . 00058 ; P ( HFD versus CTRL ; + / + ; t = 90 min ) = 0 . 0010 ; P ( HFD versus CTRL ; + / + ; t = 120 min ) = 0 . 023 ; P ( HFD versus CTRL ; − / − ; t = 60 min ) = 0 . 032 . AUC : P ( − / − versus + / + ; CTRL ) = 0 . 023 ; P ( − / − versus + / + ; HFD ) = 0 . 035 ; P ( HFD versus CTRL ; + / + ) = 0 . 042 . D Insulin tolerance test ( ITT ) was performed on 6 - h - fasted mice during 150 min after injection of 0 . 70 U insulin / kg of body weight . Curves on the left show blood glucose level monitored after injection of insulin . Histograms on the right show area above curve ( AAC ) . Fasting blood glucose : P ( − / − versus + / + ; CTRL ; t = 15 min = 0 . 012 ; P ( − / − versus + / + ; CTRL ; t = 30 min ) = 0 . 022 ; P ( − / − versus + / + ; HFD ; t = 0 min ) = 0 . 027 ; P ( − / − versus + / + ; HFD ; t = 15 min ) = 0 . 011 ; P ( − / − versus + / + ; HFD ; t = 30 min ) = 0 . 0037 ; P ( − / − versus + / + ; HFD ; t = 60 min ) = 0 . 0028 ; P ( − / − versus + / + ; HFD ; t = 90 min ) = 0 . 041 ; P ( − / − versus + / + ; HFD ; t = 120 min ) = 0 . 0032 ; P ( − / − versus + / + ; HFD ; t = 150 min ) = 0 . 0025 ; P ( HFD versus CTRL ; + / + ; t = 15 min ) = 0 . 0082 ; P ( HFD versus CTRL ; + / + ; t = 30 min ) = 0 . 033 ; P ( HFD versus CTRL ; + / + ; t = 90 min ) = 0 . 047 ; P ( HFD versus CTRL ; + / + ; t = 150 min ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ; t = 15 min ) = 0 . 026 ; P ( HFD versus CTRL ; − / − ; t = 30 min ) = 0 . 0095 ; P ( HFD versus CTRL ; − / − ; t = 60 min ) = 0 . 0031 ; P ( HFD versus CTRL ; − / − ; t = 90 min ) = 0 . 033 ; P ( HFD versus CTRL ; − / − ; t = 120 min ) = 0 . 0068 ; P ( HFD versus CTRL ; − / − ; t = 150 min ) = 0 . 0082 . AAC : P ( − / − versus + / + ; HFD ) = 0 . 030 ; P ( HFD versus CTRL ; − / − ) = 0 . 037 .

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

C Glucose tolerance test (GTT) was performed on 6-h-fasted mice during 120 min after injection of 1 g glucose/kg of body weight. Curves on the left show blood glucose level monitored after injection of glucose. Histograms on the right show area under curve (AUC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 0 min) = 0.045; P (−/− versus +/+; CTRL; t = 15 min) = 0.013; P (−/− versus +/+; CTRL; t = 30 min) = 0.016; P (−/− versus +/+; CTRL; t = 60 min) = 0.030; P (−/− versus +/+; CTRL; t = 90 min) = 0.017; P (−/− versus +/+; HFD; t = 60 min) = 0.041; P (−/− versus +/+; HFD; t = 90 min) = 0.043; P (−/− versus +/+; HFD; t = 120 min) = 0.034; P (HFD versus CTRL; +/+; t = 15 min) = 0.0076; P (HFD versus CTRL; +/+; t = 30 min) = 0.0067; P (HFD versus CTRL; +/+; t = 60 min) = 0.00058; P (HFD versus CTRL; +/+; t = 90 min) = 0.0010; P (HFD versus CTRL; +/+; t = 120 min) = 0.023; P (HFD versus CTRL; −/−; t = 60 min) = 0.032. AUC: P (−/− versus +/+; CTRL) = 0.023; P (−/− versus +/+; HFD) = 0.035; P (HFD versus CTRL; +/+) = 0.042.D Insulin tolerance test (ITT) was performed on 6-h-fasted mice during 150 min after injection of 0.70 U insulin/kg of body weight. Curves on the left show blood glucose level monitored after injection of insulin. Histograms on the right show area above curve (AAC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 15 min = 0.012; P (−/− versus +/+; CTRL; t = 30 min) = 0.022; P (−/− versus +/+; HFD; t = 0 min) = 0.027; P (−/− versus +/+; HFD; t = 15 min) = 0.011; P (−/− versus +/+; HFD; t = 30 min) = 0.0037; P (−/− versus +/+; HFD; t = 60 min) = 0.0028; P (−/− versus +/+; HFD; t = 90 min) = 0.041; P (−/− versus +/+; HFD; t = 120 min) = 0.0032; P (−/− versus +/+; HFD; t = 150 min) = 0.0025; P (HFD versus CTRL; +/+; t = 15 min) = 0.0082; P (HFD versus CTRL; +/+; t = 30 min) = 0.033; P (HFD versus CTRL; +/+; t = 90 min) = 0.047; P (HFD versus CTRL; +/+; t = 150 min) = 0.028; P (HFD versus CTRL; −/−; t = 15 min) = 0.026; P (HFD versus CTRL; −/−; t = 30 min) = 0.0095; P (HFD versus CTRL; −/−; t = 60 min) = 0.0031; P (HFD versus CTRL; −/−; t = 90 min) = 0.033; P (HFD versus CTRL; −/−; t = 120 min) = 0.0068; P (HFD versus CTRL; −/−; t = 150 min) = 0.0082. AAC: P (−/− versus +/+; HFD) = 0.030; P (HFD versus CTRL; −/−) = 0.037.


A , B ( A , B ) Immunocytofluorescent staining of TP53INP1 ( red ) and insulin ( green ) in mouse pancreatic sections ( A ) and single human islet beta cell ( B ) . Scale bars represent 50 μm ( A ) and 10 μm ( B ) .

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

A, B (A, B) Immunocytofluorescent staining of TP53INP1 (red) and insulin (green) in mouse pancreatic sections (A) and single human islet beta cell (B). Scale bars represent 50 μm (A) and 10 μm (B).


C - E Quantitative PCR for Tp53inp1 mRNA levels in tissues and cells from rat ( C ) and C57BL / 6Jmice fed with a normal diet ( 5 % fat ; ND ) or an high - fat diet ( 45 % fat ; HFD ) ( D , E ) . Results are expressed as the mean ± SEM and are representative of two independent experiments . n = 2 for ratliver , islets and mousespleen ; n = 4 for INS - 1E cells ; n = 5 for mouseexocrine pancreas ; n = 6 for Min6 cells and ND and HFD islets ; n = 11 for mouseislets . * P = 0 . 035 for HFD versus ND .

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

C-E Quantitative PCR for Tp53inp1 mRNA levels in tissues and cells from rat (C) and C57BL/6Jmice fed with a normal diet (5% fat; ND) or an high-fat diet (45% fat; HFD) (D, E). Results are expressed as the mean ± SEM and are representative of two independent experiments. n = 2 for ratliver, islets and mousespleen; n = 4 for INS-1E cells; n = 5 for mouseexocrine pancreas; n = 6 for Min6 cells and ND and HFD islets; n = 11 for mouseislets. *P = 0.035 for HFD versus ND.


Immortalized MEFs ( MEFi ) deficient ( − / − ) or not ( + / + ) for TP53INP1 were left untreated ( NT ) or treated with 3 - methyladenine ( 3 - MA , 5 mM ) during 4 h . Histograms show mt ROS level measured by flow cytometry upon MitoSox staining . P ( − / − versus + / + ; NT ) = 0 . 019 ; P ( − / − versus + / + ; 3 - MA ) = 0 . 025 ; P ( 3 - MA versus NT ; + / + ) = 0 . 031 . Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker staining . P ( − / − versus + / + ; NT ) = 0 . 012 ; P ( 3 - MA versus NT ; + / + ) = 0 . 036 . Histogram shows MitoSox fluorescence normalized with MitoTracker fluorescence . P ( − / − versus + / + ; NT ) = 0 . 041 ; P ( − / − versus + / + ; 3 - MA ) = 0 . 030 ; P ( 3 - MA versus NT ; + / + ) = 0 . 045 .

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

Immortalized MEFs (MEFi) deficient (−/−) or not (+/+) for TP53INP1 were left untreated (NT) or treated with 3-methyladenine (3-MA, 5 mM) during 4 h.Histograms show mt ROS level measured by flow cytometry upon MitoSox staining. P (−/− versus +/+; NT) = 0.019; P (−/− versus +/+; 3-MA) = 0.025; P (3-MA versus NT; +/+) = 0.031.Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker staining. P (−/− versus +/+; NT) = 0.012; P (3-MA versus NT; +/+) = 0.036.Histogram shows MitoSox fluorescence normalized with MitoTracker fluorescence. P (−/− versus +/+; NT) = 0.041; P (−/− versus +/+; 3-MA) = 0.030; P (3-MA versus NT; +/+) = 0.045.


After 4 h recovering in normal media , H2O2 ( 1 h , 100 μM ) or non - treated ( NT ) MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were observed by transmission electron microscopy ( TEM ) . N = nucleus ; white arrow = mitophagic vacuoles . Scale bar represents 0 . 5 μm . Mean size of mitochondrion ( area ) , number of mitochondria and mitophagic vacuoles normalized by cytoplasmic surface area were quantified . Size : P ( − / − versus + / + ; H2O2 ) = 0 . 000027 ; P ( H2O2 versus NT ; + / + ) = 0 . 0070 ; P ( H2O2 versus NT ; − / − ) = 0 . 013 . Nb mito . : P ( − / − versus + / + ; NT ) = 0 . 035 ; P ( − / − versus + / + ; H2O2 ) = 0 . 016 . Nb vacuoles . : P ( − / − versus + / + ; NT ) = 0 . 047 .

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

After 4 h recovering in normal media, H2O2 (1 h, 100 μM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were observed by transmission electron microscopy (TEM). N = nucleus; white arrow = mitophagic vacuoles. Scale bar represents 0.5 μm.Mean size of mitochondrion (area), number of mitochondria and mitophagic vacuoles normalized by cytoplasmic surface area were quantified. Size: P (−/− versus +/+; H2O2) = 0.000027; P (H2O2 versus NT; +/+) = 0.0070; P (H2O2 versus NT; −/−) = 0.013. Nb mito.: P (−/− versus +/+; NT) = 0.035; P (−/− versus +/+; H2O2) = 0.016. Nb vacuoles.: P (−/− versus +/+; NT) = 0.047.


After 4 h recovering in normal media , TCLs from H2O2 - ( 1 h , 100 μM ) , NAC - ( 24 h , 10 mM ) or non - treated ( NT ) MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were analyzed by immunoblotting for PGC - 1α , PINK1 , PARKIN , BNIP3 , BNIP3L / NIX , VDAC1 and β - tubulin .

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

After 4 h recovering in normal media, TCLs from H2O2- (1 h, 100 μM), NAC- (24 h, 10 mM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were analyzed by immunoblotting for PGC-1α, PINK1, PARKIN, BNIP3, BNIP3L/NIX, VDAC1 and β-tubulin.


HEK293T cells were cotransfected with plasmids encoding TP53INP1α - NTAP or TP53INP1β - NTAP . TP53INP1α - or β - NTAP was precipitated with a streptavidin - containing resin ( P ) , resolved by PAGE and Western blots developed with anti - TP53INP1 ( TP53INP1 precipitation control ) , anti - PINK1 , anti - PARKIN , anti - BNIP3 or anti - BNIP3L / NIX antibody . Western blot on TCL ( on the right ) served as a transfection control .

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

HEK293T cells were cotransfected with plasmids encoding TP53INP1α-NTAP or TP53INP1β-NTAP. TP53INP1α- or β-NTAP was precipitated with a streptavidin-containing resin (P), resolved by PAGE and Western blots developed with anti-TP53INP1 (TP53INP1 precipitation control), anti-PINK1, anti-PARKIN, anti-BNIP3 or anti-BNIP3L/NIX antibody. Western blot on TCL (on the right) served as a transfection control.


Three - month - old TP53INP1 - deficient and WT male mice were sacrificed and their livers harvested . Mitochondrial lysates ( Mito ) were purified from total liver lysates ( TCL ) , and both were analyzed by immunoblotting for TP53INP1 , PINK1 , PARKIN , VDAC1 and β - tubulin .

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

Three-month-old TP53INP1-deficient and WT male mice were sacrificed and their livers harvested. Mitochondrial lysates (Mito) were purified from total liver lysates (TCL), and both were analyzed by immunoblotting for TP53INP1, PINK1, PARKIN, VDAC1 and β-tubulin.


A - D High - resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient ( − / − , black bars ) or not ( + / + , white bars ) using glucose ( A ) , lipid - related substrates ( C ) or complex IV substrate ( D ) . High - resolution respirometry was also performed on intact MEFi ( B ) . For different respiratory state details ( routine , 3 , leak , and ETS capacity ) , please refer to the Materials and Methods section . Results are expressed as the mean ± SEM and are representative of three independent experiments . In ( A ) : P ( − / − versus + / + ; State 3 ) = 0 . 0079 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( B ) : P ( − / − versus + / + ; routine ) = 0 . 0159 ; P ( − / − versus + / + ; leak ) = 0 . 0159 ; P ( − / − versus + / + ; ETS ) = 0 . 0159 ; n = 5 in each group . In ( C ) : P ( − / − versus + / + ; State 3 ) = 0 . 029 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( D ) : P ( − / − versus + / + ; ETS ) = 0 . 0259 ; n = 5 in each group . * TP53INP1 − / − versus TP53INP1 + / + ; 1 character : P < 0 . 05 ; 2 characters : P < 0 . 01 .

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

A-D High-resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient (−/−, black bars) or not (+/+, white bars) using glucose (A), lipid-related substrates (C) or complex IV substrate (D). High-resolution respirometry was also performed on intact MEFi (B). For different respiratory state details (routine, 3, leak, and ETS capacity), please refer to the Materials and Methods section. Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A): P (−/− versus +/+; State 3) = 0.0079; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (B): P (−/− versus +/+; routine) = 0.0159; P (−/− versus +/+; leak) = 0.0159; P (−/− versus +/+; ETS) = 0.0159; n = 5 in each group. In (C): P (−/− versus +/+; State 3) = 0.029; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (D): P (−/− versus +/+; ETS) = 0.0259; n = 5 in each group. *TP53INP1−/− versus TP53INP1 +/+; 1 character: P < 0.05; 2 characters: P < 0.01.


E TCLs from MEFi were analyzed by immunoblotting for CPT1A , CPT1B and β - tubulin . Results are representative of three independent experiments .

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

E TCLs from MEFi were analyzed by immunoblotting for CPT1A, CPT1B and β-tubulin. Results are representative of three independent experiments.


MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were seeded in media containing or not 10 mM NAC . Forty - eight hours later , MEFi were treated or not ( NT ) with 100 μM hydrogen peroxide ( H2O2 ) during 1 h in serum free media . Cells were left to recover for 4 h in normal media containing or not NAC ( 10 mM ) , GW - 9662 ( PPARγ inhibitor , 10 μM ) or PNU - 74654 ( Wnt / β - catenin pathway inhibitor , 50 μM ) before being harvested . Cells were observed by TEM . Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure . Scale bar represents 0 . 5 μm . Results are expressed as the mean ± SEM and are representative of three independent experiments . * P ( − / − versus + / + ; NT ) = 0 . 00086 ; # P ( H2O2 versus NT ; + / + ) = 0 . 013 .

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

MEFi deficient (−/−) or not (+/+) for TP53INP1 were seeded in media containing or not 10 mM NAC. Forty-eight hours later, MEFi were treated or not (NT) with 100 μM hydrogen peroxide (H2O2) during 1 h in serum free media. Cells were left to recover for 4 h in normal media containing or not NAC (10 mM), GW-9662 (PPARγ inhibitor, 10 μM) or PNU-74654 (Wnt/β-catenin pathway inhibitor, 50 μM) before being harvested. Cells were observed by TEM.Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure. Scale bar represents 0.5 μm. Results are expressed as the mean ± SEM and are representative of three independent experiments. *P (−/− versus +/+; NT) = 0.00086; #P (H2O2 versus NT; +/+) = 0.013.


Cells were also observed by fluorescence microscopy after Bodipy493 / 503 staining of LD ( green ) and DAPI staining of nucleus ( blue ) . L = lipid droplets . Scale bar represents 100 μm . Results are representative of three independent experiments .

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Cells were also observed by fluorescence microscopy after Bodipy493/503 staining of LD (green) and DAPI staining of nucleus (blue). L = lipid droplets. Scale bar represents 100 μm. Results are representative of three independent experiments.


TCLs from MEFi were analyzed by immunoblotting for β - catenin , PPARγ , ATGL , MGLL and β - tubulin . Results are representative of three independent experiments .

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TCLs from MEFi were analyzed by immunoblotting for β-catenin, PPARγ, ATGL, MGLL and β-tubulin. Results are representative of three independent experiments.


( A ) Third instar larval skeletal musculature stained with Phalloidin ( F - actin ) . In this and subsequent figures , we assayed the ventral longitudinal muscles ( highlighted in green ) .

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(A) Third instar larval skeletal musculature stained with Phalloidin (F-actin). In this and subsequent figures, we assayed the ventral longitudinal muscles (highlighted in green).


( B - D ) GFP - Atg8 , overexpressed using the Dmef2 - Gal4 driver , labels autophagosomes . ( B ) , starved on low - nutrientfood for 6 h ( C ) , or starved on low - nutrientfood + 2 . 5 mg / ml CQ for 6 h ( D ) . GFP - Atg8 - labeled vesicles appeared only in the starved animals ( C - D ) , localizing around the nucleus and between myofibers . ( D ) CQ treatment caused accumulation of bloated GFP - Atg8 - labeled vesicles .

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(B-D) GFP-Atg8, overexpressed using the Dmef2-Gal4 driver, labels autophagosomes. (B), starved on low-nutrientfood for 6 h (C), or starved on low-nutrientfood +2.5 mg/ml CQ for 6 h (D). GFP-Atg8-labeled vesicles appeared only in the starved animals (C-D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP-Atg8-labeled vesicles.


( E - G ) Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - HRP - Lamp1 animals were assayed for Lamp1 and Atg8 localization ( anti - HRP , red ; GFP , green ; DAPI , blue ) . . ( E ) High - nutrient food suppressed formation of both GFP - Atg8 and HRP - Lamp1 - labeled vesicles . ( F ) Colocalization of GFP - Atg8 and HRP - Lamp in animals starved on low - nutrient food . The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp . ( G ) Addition of CQ to the starvation diet resulted in accumulation of both GFP - Atg8 and HRP - Lamp - labeled vesicles , but they failed to colocalize .

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(E-G) Dmef2-Gal4, UAS-GFP-Atg8/UAS-HRP-Lamp1 animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP-Atg8 and HRP-Lamp1-labeled vesicles. (F) Colocalization of GFP-Atg8 and HRP-Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP-Atg8 and HRP-Lamp-labeled vesicles, but they failed to colocalize.


( H ) Quantification of the number of GFP - Atg8 , HRP - Lamp1 , or GFP - Atg8 + HRP - Lamp1 vesicles in starved or starved + CQ muscles .

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(H) Quantification of the number of GFP-Atg8, HRP-Lamp1, or GFP-Atg8+HRP-Lamp1 vesicles in starved or starved +CQ muscles.


( I - M ) The core Atg genes are required for starvation - induced autophagy in both wild - type and CQ - treated skeletal muscles . Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1 larvae were starved on low - nutrient food for 6 h ( I ) or starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h ( J ) . Note that Atg1 knockdown completely abolished the formation of GFP - Atg8 - labeled autophagosomes ( compare I - J to C - D ) . ( K - L ) Dmef2 - Gal4 , UAS - GFP - Atg8 , Atg1Δ3d larvae failed to form GFP - Atg8 vesicles when starved or starved and treated with CQ . ( M ) Quantification of autophagy changes due to Atg gene knockdown in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . Each of the 10 UAS - Atg RNAi transgenes tested caused a highly significant decrease ( p < . 01 ) in the total area of GFP - Atg8vesicles . SEM is indicated , with n = 5 ventral longitudinal muscles from individual animals .

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(I-M) The core Atg genes are required for starvation-induced autophagy in both wild-type and CQ-treated skeletal muscles. Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1 larvae were starved on low-nutrient food for 6 h (I) or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (J). Note that Atg1 knockdown completely abolished the formation of GFP-Atg8-labeled autophagosomes (compare I-J to C-D). (K-L) Dmef2-Gal4, UAS-GFP-Atg8, Atg1Δ3d larvae failed to form GFP-Atg8 vesicles when starved or starved and treated with CQ. (M) Quantification of autophagy changes due to Atg gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. Each of the 10 UAS-Atg RNAi transgenes tested caused a highly significant decrease (p<.01) in the total area of GFP-Atg8vesicles. SEM is indicated, with n = 5 ventral longitudinal muscles from individual animals.


. ( N - O ) EM of muscles from Dmef2 - Gal4 , UAS - whitei larvae . Animals starved on low - nutrient food + 2 . 5 mg / ml CQ ( O ) accumulated vesicles in the intermyofibril spaces ( red asterisk ) , disrupting the integrity of the sarcomere compared to non - CQ - treated control muscles ( N ) .

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. (N-O) EM of muscles from Dmef2-Gal4, UAS-whitei larvae. Animals starved on low-nutrient food +2.5 mg/ml CQ (O) accumulated vesicles in the intermyofibril spaces (red asterisk), disrupting the integrity of the sarcomere compared to non-CQ-treated control muscles (N).


( P ) CQ treatment increased the larval crawling time of Dmef2 - Gal4 , UAS - whitei larvae in starved animals , and weakly in fed animals . ( Q ) CQ treatment increased the larval righting time of Dmef2 - Gal4 , UAS - whitei larvae in starved but not fed animals . For both locomotor assays , SEM is indicated for n = 10 larvae ( * p < . 05 , * * p < . 01 ) .

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(P) CQ treatment increased the larval crawling time of Dmef2-Gal4, UAS-whitei larvae in starved animals, and weakly in fed animals. (Q) CQ treatment increased the larval righting time of Dmef2-Gal4, UAS-whitei larvae in starved but not fed animals. For both locomotor assays, SEM is indicated for n = 10 larvae (*p<.05, **p<.01).


( A ) Sectioned third instar OreR larva stained with Periodic acid - Schiff ( PAS ) . The muscles , but not the fat body , are stained purple , indicating high levels of glycogen ( m , muscle ; bw , body wall ; fb , fat body ) .

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(A) Sectioned third instar OreR larva stained with Periodic acid-Schiff (PAS). The muscles, but not the fat body, are stained purple, indicating high levels of glycogen (m, muscle; bw, body wall; fb, fat body).


( B ) Glycogen was also detected in muscle from Dmef2 - Gal4 , UAS - GFP - Atg8 larvae , immunostained with an antiglycogen monoclonal antibody .

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(B) Glycogen was also detected in muscle from Dmef2-Gal4, UAS-GFP-Atg8 larvae, immunostained with an antiglycogen monoclonal antibody.


( C ) GFP - Atg8 vesicles colocalized with glycogen in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h ( GFP , green ; antiglycogen , red ) .

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(C) GFP-Atg8 vesicles colocalized with glycogen in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h (GFP, green; antiglycogen, red).


D ) HRP - Lamp1 vesicles show less colocalization with glycogen in UAS - HRP - Lamp1 ; Dmef2 - Gal4 larvae starved and treated with CQ .

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D) HRP-Lamp1 vesicles show less colocalization with glycogen in UAS-HRP-Lamp1;Dmef2-Gal4 larvae starved and treated with CQ.


( E ) Quantification of GFP - Atg8 or HRP - Lamp1 vesicles with glycogen .

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(E) Quantification of GFP-Atg8 or HRP-Lamp1 vesicles with glycogen.


( F - G ) EM from Dmef2 - Gal4 , UAS - whitei larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . ( F ) Double - and single - membrane vesicles containing glycogen granules accumulated between myofibers ( s , sarcomere ; m , mitochondrion ; AVs , autophagic vesicles ) . ( G ) Higher magnification view of region outlined in ( E ) . ( H ) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2 - Gal4 , UAS - whitei larva starved on low - nutrient food for 6 h . Arrow points to double membrane .

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(F-G) EM from Dmef2-Gal4, UAS-whitei larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Double- and single-membrane vesicles containing glycogen granules accumulated between myofibers (s, sarcomere; m, mitochondrion; AVs, autophagic vesicles). (G) Higher magnification view of region outlined in (E). (H) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2-Gal4, UAS-whitei larva starved on low-nutrient food for 6 h. Arrow points to double membrane.


( A - D ) Time course of autophagy induction in Dmef2 - Gal4 , UAS - GFP - Atg8 muscles , accompanied by quantification of GFP - Atg8 and glycogen colocalization . Animals were fed for 18 h in high - nutrient food + 2 . 5 mg / ml CQ , then starved on low - nutrient food + 2 . 5 mg / ml CQ for 0 - 8 h ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( A ) At time point 0 , following 18 h in high - nutrient food + CQ , the muscles contained large amounts of glycogen with no apparent autophagy . ( B ) At 3 h of starvation , glycogen stores were still high , and GFP - Atg8 - labeled vesicles began to appear . ( C - D ) At 6 and 8 h of starvation , the majority of GFP - Atg8 - labeled vesicles colocalized with glycogen .

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(A-D) Time course of autophagy induction in Dmef2-Gal4, UAS-GFP-Atg8 muscles, accompanied by quantification of GFP-Atg8 and glycogen colocalization. Animals were fed for 18 h in high-nutrient food +2.5 mg/ml CQ, then starved on low-nutrient food +2.5 mg/ml CQ for 0-8 h (antiglycogen, red; GFP, green; DAPI, blue). (A) At time point 0, following 18 h in high-nutrient food +CQ, the muscles contained large amounts of glycogen with no apparent autophagy. (B) At 3 h of starvation, glycogen stores were still high, and GFP-Atg8-labeled vesicles began to appear. (C-D) At 6 and 8 h of starvation, the majority of GFP-Atg8-labeled vesicles colocalized with glycogen.


( E ) Time course of glycogen levels in Dmef2 - Gal4 carcasses ( muscle + body wall ) . Animals were fed for 24 h in high - nutrient food , then starved on low - nutrient food + / − 2 . 5 mg / ml CQ for 0 - 24 h . Starvation caused reduction of glycogen levels in both untreated and CQ - treated larvae over time . However , after 6 h of starvation , CQ treatment significantly increased glycogen levels compared to controls . SEM is indicated for n = 5 - 8 samples ( * p < . 05 , * * p < . 01 ) .

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(E) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall). Animals were fed for 24 h in high-nutrient food, then starved on low-nutrient food +/− 2.5 mg/ml CQ for 0-24 h. Starvation caused reduction of glycogen levels in both untreated and CQ-treated larvae over time. However, after 6 h of starvation, CQ treatment significantly increased glycogen levels compared to controls. SEM is indicated for n = 5-8 samples (*p<.05, **p<.01).


( F - G ) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . ( F ) Autophagy levels were high in control Dmef2 - Gal4 / UAS - whitei larvae . Muscles from ( G ) UAS - Rheb / + ; Dmef2 - Gal4 / + , ( H ) Dmef2 - Gal4 / UAS - Tsc1i , and ( I ) Dmef2 - Gal4 / UAS - gigi all failed to induce autophagy .

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(F-G) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Autophagy levels were high in control Dmef2-Gal4/UAS-whitei larvae. Muscles from (G) UAS-Rheb/+; Dmef2-Gal4/+, (H) Dmef2-Gal4/UAS-Tsc1i, and (I) Dmef2-Gal4/UAS-gigi all failed to induce autophagy.


( A - B ) Glycogen phosphorylase is not required for glycogen autophagy ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( A ) UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h exhibited high levels of colocalization between GFP - Atg8 and glycogen . ( B ) Higher magnification of region outlined in ( A )

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(A-B) Glycogen phosphorylase is not required for glycogen autophagy (antiglycogen, red; GFP, green; DAPI, blue). (A) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h exhibited high levels of colocalization between GFP-Atg8 and glycogen.(B) Higher magnification of region outlined in (A)


C - F ) Dmef2 - Gal4 , UAS - GFP - Atg8 larvae with GlyP and / or Atg1 knockdown were fed on high - nutrient food for 18 h before being starved on low - nutrient food ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( C ) UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + larval muscle contained high levels of glycogen prior to starvation , indicating no defect in glycogen synthesis . ( D ) Following 24 h starvation UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + muscles contained no glycogen detectable by antibody staining . ( E ) Following 24 h of starvation Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i muscles contained no glycogen . ( F ) Double - mutant larvae UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i larval muscles contained high levels of glycogen after 24 h of starvation , indicating an inability to break down glycogen .

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C-F) Dmef2-Gal4, UAS-GFP-Atg8 larvae with GlyP and/or Atg1 knockdown were fed on high-nutrient food for 18 h before being starved on low-nutrient food (antiglycogen, red; GFP, green; DAPI, blue). (C) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larval muscle contained high levels of glycogen prior to starvation, indicating no defect in glycogen synthesis. (D) Following 24 h starvation UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ muscles contained no glycogen detectable by antibody staining. (E) Following 24 h of starvation Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i muscles contained no glycogen. (F) Double-mutant larvae UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i larval muscles contained high levels of glycogen after 24 h of starvation, indicating an inability to break down glycogen.


( G ) Time course of glycogen levels in Dmef2 - Gal4 carcasses ( muscle + body wall ) with expression of UAS - RNAi transgenes targeting white , GlyP , Atg1 , or GlyP + Atg1 . Simultaneous knockdown of GlyP and Atg1 , but not either gene alone , significantly reduced glycogen degradation compared to the white control after 24 h of starvation , consistent with immunostaining ( C - F ) . Between 6 and 12 h of starvation , individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels , indicating a reduced rate of glycogen degradation . SEM is indicated for n = 5 - 8 samples . The p values were calculated relative to white RNAi control at each time point ( * p < . 05 , * * p < . 01 ) .

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(G) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall) with expression of UAS-RNAi transgenes targeting white, GlyP, Atg1, or GlyP+Atg1. Simultaneous knockdown of GlyP and Atg1, but not either gene alone, significantly reduced glycogen degradation compared to the white control after 24 h of starvation, consistent with immunostaining (C-F). Between 6 and 12 h of starvation, individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels, indicating a reduced rate of glycogen degradation. SEM is indicated for n = 5-8 samples. The p values were calculated relative to white RNAi control at each time point (*p<.05, **p<.01).


( A - D ) Glycogen synthase ( GlyS ) is required for glycogen synthesis in D . melanogaster muscles . PAS staining for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi muscles ( B ) compared to control Dmef2 - Gal4 / UAS - whitei muscles ( A ) . Antiglycogen immunostaining for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi muscles ( D ) compared to Dmef2 - Gal4 / UAS - whitei control muscles ( C ) .

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(A-D) Glycogen synthase (GlyS) is required for glycogen synthesis in D. melanogaster muscles. PAS staining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (B) compared to control Dmef2-Gal4/UAS-whitei muscles (A). Antiglycogen immunostaining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (D) compared to Dmef2-Gal4/UAS-whitei control muscles (C).


( C ) . ( E - K ) GlyS is required for the formation of large CQ - induced autophagosomes . Vesicles are much smaller in Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - GlySi larval muscle starved 6 h in low - nutrient food + 2 . 5 mg / ml CQ ( G ) than in control Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - whitei larval muscle ( E ) . ( F , H ) The difference in autophagosome size is clearly evident at high magnification .

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(C). (E-K) GlyS is required for the formation of large CQ-induced autophagosomes. Vesicles are much smaller in Dmef2-Gal4, UAS-GFP-Atg8/UAS-GlySi larval muscle starved 6 h in low-nutrient food +2.5 mg/ml CQ (G) than in control Dmef2-Gal4, UAS-GFP-Atg8/UAS-whitei larval muscle (E). (F, H) The difference in autophagosome size is clearly evident at high magnification.


( I - K ) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . SEM is indicated , with n = 5 ( I ) or n = 10 ( J - K ) ventral longitudinal muscles from individual animals ( * p < . 05 , * * p < . 01 ) . ( I ) Each of the four UAS - GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP - Atg8vesicles in the muscle compared to the UAS - whitei control . ( J ) Vesicle number was unchanged by GlyS knockdown . ( K ) UAS - GlyS RNAi caused a highly significant decrease in the mean vesicle size ( area ) compared to the control .

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(I-K) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. SEM is indicated, with n = 5 (I) or n = 10 (J-K) ventral longitudinal muscles from individual animals (*p<.05, **p<.01). (I) Each of the four UAS-GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP-Atg8vesicles in the muscle compared to the UAS-whitei control. (J) Vesicle number was unchanged by GlyS knockdown. (K) UAS-GlyS RNAi caused a highly significant decrease in the mean vesicle size (area) compared to the control.


( L - M ) GlyS gene expression , monitored using a MiMIC transposon insertion ( MI01490 ) , showed expression in the larval muscle ( L ) but not the fat body ( M ) ( green , GFP ; blue , DAPI ) .

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(L-M) GlyS gene expression, monitored using a MiMIC transposon insertion (MI01490), showed expression in the larval muscle (L) but not the fat body (M) (green, GFP; blue, DAPI).


N - O ) Larvae were starved on low - nutrient food for 6 h prior to dissection of the fat bodies . Autophagy in Cg - Gal4 / + ; UAS - GFP - Atg8 / UAS - GlySi ( O ) was not substantially different from autophagy in Cg - Gal4 / + ; UAS - GFP - Atg8 / UAS - whitei control fat bodies ( GFP , green ; DAPI , blue ) .

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N-O) Larvae were starved on low-nutrient food for 6 h prior to dissection of the fat bodies. Autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-GlySi (O) was not substantially different from autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-whitei control fat bodies (GFP, green; DAPI, blue).


. ( P ) EM of muscle from Dmef2 - Gal4 / UAS - GlySi animal starved on low - nutrient food + CQ . Note that the intermyofibril spaces ( red asterisk ) and sarcomere structure are not distorted .

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. (P) EM of muscle from Dmef2-Gal4/UAS-GlySi animal starved on low-nutrient food +CQ. Note that the intermyofibril spaces (red asterisk) and sarcomere structure are not distorted.


Q ) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h . SEM is indicated for n = 10 larvae . The p values were calculated relative to white RNAi control larvae ( * p < . 05 , * * p < . 01 ) .

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Q) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h. SEM is indicated for n = 10 larvae. The p values were calculated relative to white RNAi control larvae (*p<.05, **p<.01).