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

(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 : of unwinding experiments showing of free relative to R - loop substrate ( unwound product ) as a function of DDX5 alone ( black ) or in presence of BRCA2 ( red ) or BRCA2LT3 ( blue ) or BRCA2T1 ( pink ) . data represent mean ± SD of at least three independent experiments . Right : SDS - PAGE showing 500 ng of purified 2XMBP - BRCA2LT3 150 ng of purified BRCA2 used in unwinding .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 in which purified MBP - DDX5 - GST ( 50 - 200 nM ) was incubated with R - loop synthetic substrate [ γ32P ] ATP in presence or absence of or 6 nM purified BRCA2LT3 . Right : of ATP hydrolysed in each condition . No control was used as background . data represent 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 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

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

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

(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 siC level at each locus ) at MALAT1 , RRPH1 HIST1H2BG loci in DLD1 BRCA2 - / - bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . data represent mean ± from seven independent experiments . statistical significance of difference was calculated with paired t - Student ; p - values show 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

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

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

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

(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 of in situ PLA performed between S9 . 6 ( - hybrids ) anti - γH2AX antibodies in BRCA2 deficient DLD1 ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . When indicated , 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 are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on DAPI image ( Image J ) our outlined in yellow . Bottom : of number of PLA spots per nucleus . At least 600 were counted per condition from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median 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 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

(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 : of unwinding experiments showing of free relative to R - loop substrate ( unwound product ) as a function of DDX5 alone ( black ) or in presence of BRCA2T1 - T207A ( green ) . data represent mean ± SD of at least three independent experiments . Bottom right : SDS - PAGE showing 650 ng of purified 2XMBP - BRCA2T1 - T207A used in unwinding .

(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 of stained for RAD51 ( green ) in U2OS depleted of DDX5 ( siDDX5 ) in control ( siC ) in non - treated ( - ) or different time points after exposure to IR ( 6Gy ) , as indicated . Scale bar indicates 10 µm . Bottom : Graph showing average number of RAD51 repair foci in both lines . data represent mean ± 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

(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 of DLD1 BRCA2 deficient ( BRCA2 - / - ) or BRCA2 - / - 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 average number of RAD51 repair foci in three lines . data represent mean ± 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

(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 of DLD1 BRCA2 WT or BRCA2 - T207A 4h post - irradiation ( 6Gy ) hybridized with anti - γH2AX anti - RPA antibodies . Right : of number of γH2AX RPA foci per nucleus , as indicated . data represent at least 400 per condition from two independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median 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

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

(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 fraction . When indicated , were irradiated ( 2h treatment ) / or transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . α - tubulin Histone H3 signals are shown as markers of fraction respectively . Bottom : of RAD51 detected by Western blot with a RAD51 specific antibody in fraction relative to RAD51 in WCE . Results are presented as fold change compared to untreated sample . data represents mean ± SD of three independent experiments . Statistical significance of difference was calculated with unpaired t p - values show 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

(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 ( − / − ) WT ( + / + ) mice were subjected to a high - fat diet ( HFD , 60 % fat ) or a control diet ( CTRL ) for 16 . Mice drank tap water or tap water supplemented with NAC ( 10 mg / ml or 1 % ) . Curves show mice 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 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

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 end of protocol , mice were sacrificed ; liver epididymal renal fat masses were taken weighed . Histograms show organ . 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 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 ) of 6 - h - fasted mice at beginning ( Week 0 ) / or at end of 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 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 ( GTT ) was performed on 6 - h - fasted mice during 120 min after injection of 1 g glucose / kg of . Curves on left show blood glucose level monitored after injection of glucose . Histograms on right show 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 ( ITT ) was performed on 6 - h - fasted mice during 150 min after injection of 0 . 70 U insulin / kg of . Curves on left show blood glucose level monitored after injection of insulin . Histograms on right show 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 B-MEASURED_VAR 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 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 B-CONTROLLED_VAR 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 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 of TP53INP1 ( red ) insulin ( green ) in mouse pancreatic sections ( A ) single human islet beta ( B ) . Scale bars represent 50 μm ( A ) 10 μm ( B ) .

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

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 in tissues from rat ( C ) C57BL / 6Jmice fed with a normal diet ( 5 % fat ; ND ) or an high - fat diet ( 45 % fat ; HFD ) ( D , E ) . Results are expressed as mean ± are representative of two independent experiments . n = 2 for ratliver , islets mousespleen ; n = 4 for INS - 1E ; n = 5 for mouseexocrine pancreas ; n = 6 for Min6 ND 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

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 . 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 . P ( − / − versus + / + ; NT ) = 0 . 012 ; P ( 3 - MA versus NT ; + / + ) = 0 . 036 . Histogram shows MitoSox normalized with MitoTracker . 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 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 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 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 ( ) . N = nucleus ; arrow = mitophagic vacuoles . Scale bar represents 0 . 5 μm . Mean of mitochondrion ( ) , number of mitochondria mitophagic vacuoles normalized by surface were quantified . : 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 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 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

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 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 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 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

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

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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 ( + / + , bars ) using glucose ( A ) , lipid - related substrates ( C ) or IV substrate ( D ) . High - resolution respirometry was also performed on intact MEFi ( B ) . For different respiratory state details ( routine , 3 , leak , ETS capacity ) , please refer to Materials Methods section . Results are expressed as mean ± 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 .

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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 β - tubulin . Results are representative of three independent experiments .

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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 . 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 . were observed by . Number of LD normalized by surface is shown in both histograms at bottom of figure . Scale bar represents 0 . 5 μm . Results are expressed as mean ± are representative of three independent experiments . * P ( − / − versus + / + ; NT ) = 0 . 00086 ; # P ( H2O2 versus NT ; + / + ) = 0 . 013 .

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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.


were also observed by microscopy after Bodipy493 / 503 of LD ( green ) DAPI 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 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 subsequent figures , we assayed ventral longitudinal ( 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 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 starved ( C - D ) , localizing around nucleus 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 were assayed for Lamp1 Atg8 localization ( anti - HRP , red ; GFP , green ; DAPI , blue ) . . ( E ) High - nutrient food suppressed formation of both GFP - Atg8 HRP - Lamp1 - labeled vesicles . ( F ) Colocalization of GFP - Atg8 HRP - Lamp in starved on low - nutrient food . yellow arrowhead points to a vesicle positive for both Atg8 Lamp . ( G ) Addition of CQ to starvation diet resulted in accumulation of both GFP - Atg8 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 ) of number of GFP - Atg8 , HRP - Lamp1 , or GFP - Atg8 + HRP - Lamp1 vesicles in starved or starved + CQ .

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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 ) Atg genes are required for starvation - induced autophagy in both wild - type CQ - treated skeletal . 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 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 treated with CQ . ( M ) 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 10 UAS - Atg RNAi transgenes tested caused a highly significant decrease ( p < . 01 ) in total of GFP - Atg8vesicles . is indicated , with n = 5 ventral longitudinal from .

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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 from Dmef2 - Gal4 , UAS - whitei larvae . starved on low - nutrient food + 2 . 5 mg / ml CQ ( O ) accumulated vesicles in intermyofibril spaces ( red asterisk ) , disrupting integrity of sarcomere compared to non - CQ - treated control ( 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 larval crawling time of Dmef2 - Gal4 , UAS - whitei larvae in starved , weakly in fed . ( Q ) CQ treatment increased larval righting time of Dmef2 - Gal4 , UAS - whitei larvae in starved but not fed . For both locomotor assays , 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 ) . , but not fat , are stained purple , indicating high of glycogen ( m , muscle ; bw , wall ; fb , fat ) .

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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 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 ) 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 - single - 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 .

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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 , accompanied by of GFP - Atg8 glycogen colocalization . 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 , contained large amounts of glycogen with no apparent autophagy . ( B ) At 3 h of starvation , glycogen stores were still high , GFP - Atg8 - labeled vesicles began to appear . ( C - D ) At 6 8 h of starvation , 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 in Dmef2 - Gal4 carcasses ( muscle + wall ) . 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 in both untreated CQ - treated larvae over time . However , after 6 h of starvation , CQ treatment significantly increased glycogen compared to controls . 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 Tor pathway blocked autophagy in from larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . ( F ) Autophagy were high in control Dmef2 - Gal4 / UAS - whitei larvae . from ( G ) UAS - Rheb / + ; Dmef2 - Gal4 / + , ( H ) Dmef2 - Gal4 / UAS - Tsc1i , ( I ) Dmef2 - Gal4 / UAS - gigi 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 of colocalization between GFP - Atg8 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 / 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 of glycogen prior to starvation , indicating no defect in glycogen synthesis . ( D ) Following 24 h starvation UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + contained no glycogen detectable by antibody . ( E ) Following 24 h of starvation Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i contained no glycogen . ( F ) Double - mutant larvae UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i larval contained high 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 in Dmef2 - Gal4 carcasses ( muscle + wall ) with expression of UAS - RNAi transgenes targeting , GlyP , Atg1 , or GlyP + Atg1 . Simultaneous knockdown of GlyP Atg1 , but not either gene alone , significantly reduced glycogen degradation compared to control after 24 h of starvation , consistent with immunostaining ( C - F ) . Between 6 12 h of starvation , knockdown of GlyP or Atg1 caused a significant increase in glycogen , indicating a reduced rate of glycogen degradation . is indicated for n = 5 - 8 samples . p values were calculated relative to 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 . PAS for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi ( B ) compared to control Dmef2 - Gal4 / UAS - whitei ( A ) . Antiglycogen immunostaining for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi ( D ) compared to Dmef2 - Gal4 / UAS - whitei control ( 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 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 ) difference in autophagosome 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 ) 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 . is indicated , with n = 5 ( I ) or n = 10 ( J - K ) ventral longitudinal from ( * p < . 05 , * * p < . 01 ) . ( I ) Each of four UAS - GlyS RNAi transgenes tested caused a significant decrease in total of GFP - Atg8vesicles in muscle compared to UAS - whitei control . ( J ) Vesicle number was unchanged by GlyS knockdown . ( K ) UAS - GlyS RNAi caused a highly significant decrease in mean vesicle ( ) compared to 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 larval muscle ( L ) but not fat ( 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 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 starved on low - nutrient food + CQ . Note that intermyofibril spaces ( red asterisk ) 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 crawling time of larvae treated with CQ starved for 6 h . is indicated for n = 10 larvae . p values were calculated relative to 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).


( A ) alignment of C - terminal region of D . melanogaster , human , yeast Glycogen synthases . Identical residues are blue ; other residues are red . Conserved in three species , R593 , W609 , S651 are underlined .

(A) Protein sequence alignment of the C-terminal region of D. melanogaster, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined.


( B ) Western blot / Co - immunoprecipitation ( co - IP ) showing that Flag - Atg8 binds to a Venus - GlyS or Venus - GlyS ( S651A ) in response to starvation . Flag - Atg8 is unable to co - IP with either Venus - GlyS ( R593A ) or Venus - GlyS ( W609A ) . Venus - GlyS Venus - GlyS mutants were co - IP ' d from muscle lysate from Dmef2 - Gal4 / UAS - Flag - Atg8 or UAS - Venus - GlyS ( WT or mutant ) / + ; Dmef2 - Gal4 / UAS - Flag - Atg8third instar larvae . These were fed on high - nutrientfood for 18 h , then transferred to fresh high - nutrientfood or low - nutrientfood for 6 h .

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(B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag-Atg8 binds to a Venus-GlyS or Venus-GlyS (S651A) protein complex in response to starvation. Flag-Atg8 is unable to co-IP with either Venus-GlyS (R593A) or Venus-GlyS (W609A). Venus-GlyS and Venus-GlyS mutants were co-IP'd from muscle lysate from Dmef2-Gal4/UAS-Flag-Atg8 or UAS-Venus-GlyS(WT or mutant)/+;Dmef2-Gal4/UAS-Flag-Atg8third instar larvae. These were fed on high-nutrientfood for 18 h, and then transferred to fresh high-nutrientfood or low-nutrientfood for 6 h.


( C ) Venus - GlyS was localized predominantly to Flag - labeled autophagosomes , with weak in of cytoplasm . ( D ) Venus - GlyS ( R593A ) was found throughout cytoplasm did not colocalize with autophagosomes . ( E ) Venus - GlyS ( S651A ) was localized to autophagosomes . ( F ) Venus - GlyS ( W609A ) did not colocalize with Flag - labeled autophagosomes .

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(C) Venus-GlyS was localized predominantly to the Flag-labeled autophagosomes, with weak staining in the rest of the cytoplasm. (D) Venus-GlyS (R593A) was found throughout the cytoplasm and did not colocalize with autophagosomes. (E) Venus-GlyS (S651A) was localized to the autophagosomes. (F) Venus-GlyS (W609A) did not colocalize with the Flag-labeled autophagosomes.


( B ) Lignin accumulation patterns at endodermal surface or median positions with or without 100 nM CIF2 ( Casparian strip integrity factor 2 ) ligand treatment . Lignin cellulosic ( unmodified ) walls are stained with Basic Fuchsin Calcofluor , shown in magenta respectively . Schematics are indicating position of optical sections in a 3D illustration . For each condition , at least 10 roots were tested showed similar results in two independent experiments . arrows indicate sites of excess lignification on cortex facing ( outer ) side . Scale Bar = 5 µm . Data information : " inner " designates stele - facing endodermal surface , " outer " , cortex - facing surface .

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(B) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex facing (outer) side. Scale Bar = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( C , D ) Localization of SGN1 - Citrine lignin deposition patterns in pCASP1 : : SGN1 - Citrine lines in wild - type ( Col ) different mutant backgrounds ( sgn1 , sgn3 , cif1 cif2 ) ( C ) . myrpalm - SGN1 - Citrine localization lignin deposition patterns in pCASP1 : : myrpalm - SGN1 - Citrine lines ( D ) . Lignin ( Basic Fuchsin ) walls ( Calcoflour ) are shown in magenta respectively . For this experiment , two or three independent lines were tested . From each transgenic , 2 positions from 12 roots were observed representative pictures are shown in figure . Schematics are indicating position of optical sections in a 3D illustration . arrows in ( D ) highlight excess lignification on pericycle - facing ( inner ) side . Scale bars = 5 µm . Data information : " inner " designates stele - facing endodermal surface , " outer " , cortex - facing surface .

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

(C,D) Localization of SGN1-Citrine and lignin deposition patterns in pCASP1::SGN1-Citrine lines in wild-type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm-SGN1-Citrine localization and lignin deposition patterns in pCASP1::myrpalm-SGN1-Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcoflour White) are shown in magenta and white respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle-facing (inner) side. Scale bars = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( A ) of defects in CSD formation as number of holes per 100 µm in CASP1 - GFP domain at around 10 after onset of CASP1 - GFP expression in 5 - day - old seedlings . In box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper lower whiskers extend to maximum or minimum values no further than 1 . 5 times IQR ( interquartile range , between first third quartiles ) . One - way ANOVA was performed followed by Tukey ' s . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA Tukey ' s , 12 roots in total were observed for each condition in two independent assays ) .

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(A) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1-GFP domain at around 10 cells after onset of CASP1-GFP expression in 5-day-old seedlings. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to maximum or minimum values no further than 1.5 times IQR (interquartile range, the distance between the first and third quartiles). One-way ANOVA was performed followed by Tukey's test. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test, 12 roots in total were observed for each condition in two independent assays).


( B ) Propidium iodide ( PI ) penetration in presence or absence of CIF2 . CS barrier function was scored as exclusion of PI signal from inner side of endodermal . In box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper lower whiskers extend to maximum or minimum values no further than 1 . 5 times IQR . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA Tukey ' s . During two independent experiments , 10 roots in total were tested for each condition ) .

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(B) Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition).


( C ) [ γ - 32P ] ATP radioactive in vitro kinase of SGN3 kinase domain against SGN1 . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence equal loading of recombinant . Note that a kinase - dead SGN1 variant was used to avoid auto - phosphorylation activity of SGN1 . Also note that trigger factor represents a very big tag , accounting for high migration of TF - SGN1 . Representative result of three independent experiments is shown .

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(C) [γ-32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase-dead SGN1 variant was used to avoid auto-phosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF-SGN1. Representative result of three independent experiments is shown.


( A ) Localization of Citrine - RBOHD ( left ) or Citrine - RBOHF ( right ) in endodermal ( en ) . Both were expressed under control of pCASP1 , an endodermis - specific promoter . Representative pictures are shown , 2 positions from 10 roots for each transgenic were inspected . Scale bar = 5 µm .

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(A) Localization of Citrine-RBOHD (left) or Citrine-RBOHF (right) in endodermal (en) cells. Both proteins were expressed under the control of pCASP1, an endodermis-specific promoter. Representative pictures are shown, 2 positions from 10 roots for each transgenic line were inspected. Scale bar = 5 µm.


( B ) Lignin accumulation in WT rbohD rbohF single mutants a double mutant with or without 2 - hour 100 nM CIF2 peptide treatment . Arrowheads indicate excess lignification . Pictures are shown as overviews ( maximum projection ) or median sections . Lignin walls are shown with magenta ( stained with Basic Fuchsin ) gray ( stained with Calcofluor ) respectively . Representative pictures are shown , 12 roots ( overview ) 2 positions in 12 roots ( median section ) were inspected . Scale bars = 20 µm ( lignin overviews ) , 5 µm ( median sections ) . " inner " designates stele - facing endodermal surface , " outer " , cortex - facing surface .

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(B) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2-hour 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White) respectively. Representative pictures are shown, 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 µm (lignin overviews), 5 µm (median sections). "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( A ) Co - visualization of pCASP1 : : SGN1 - mCherry pCASP1 : : SGN3 - GFP . Note that their respective localization at PM has only restricted overlap at cortical side of CS domain ( Arrow ) . Representative picture is shown , 3 - 6 roots in 4 different transgenic lines were inspected . Scale bar = 10 µm . " inner " designates stele - facing endodermal surface , " outer " , cortex - facing surface .

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(A) Co-visualization of pCASP1::SGN1-mCherry and pCASP1::SGN3-GFP. Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain (Arrow). Representative picture is shown, 3-6 roots in 4 different transgenic lines were inspected. Scale bar = 10 µm. "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( B , C ) Overview of endodermal after with or without 1 µM CIF2 treatment for 24 hours . Red arrowheads are indicating ROS production sites boxes in dotted lines are corresponding to regions in ( sky blue creme boxes in C black boxes in D ) . Similar patterns were obtained in 39 or 41 from 5 roots with or without peptide treatment . Scale bars = 500 nm .

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(B,C) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours. Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in (sky blue and creme boxes in C and black boxes in D). Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment. Scale bars = 500 nm.


( D ) In situ H2O2 detection at Casparian strips in WT , sgn3 , sgn1 , rbohF , rbohD rbohDF with or without 24 - hour treatment of 1 µM CIF2 . Brackets arrowheads indicate Casparian strips ( seen as uniformly whitish wall areas ) H2O2 production sites ( black ) respectively . Scale bar = 1000 nm .

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(D) In situ H2O2 detection at Casparian strips in WT, sgn3, sgn1, rbohF, rbohD and rbohDF with or without 24-hour treatment of 1 µM CIF2. Brackets and arrowheads indicate Casparian strips (seen as uniformly whitish cell wall areas) and H2O2 production sites (black area) respectively. Scale bar = 1000 nm.


( E ) of ROS production as a number of pixels ( n = 23 - 44 sites from 5 roots of each condition in one experiment . ) . In box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper lower whiskers extend to maximum or minimum values no further than 1 . 5 times IQR . Different letters mean significant statistical differences . ( p < 0 . 01 , One - way ANOVA Tukey ' s . )

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(E) Quantification of ROS production as a number of dark pixels area (n = 23 - 44 sites from 5 roots of each condition in one experiment.). In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters mean significant statistical differences. (p<0.01, One-way ANOVA and Tukey's test.)


( A ) [ γ - 32P ] ATP radioactive in vitro kinase of TF - SGN1 against GST - N - terminal domains of RBOHD or F . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence equal loading of recombinant . Experiments were done independently three times with similar results .

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(A) [γ-32P]ATP radioactive in vitro kinase assay of TF-SGN1 against GST-N-terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results.


( B ) HEK293Tcell NOX activation . were transfected with indicated plasmid combinations . phosphatase inhibitor Calyculin A was added directly before start of measurements . Each data point represents mean of six wells analyzed in parallel , bars indicate S . D . . Experiments were repeated three

O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293Tcell based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel, bars indicate S.D.. Experiments were repeated three times


( A ) CASP1 - GFP lignin deposition in WT , sgn3 , rbohD , rbohF rbohDF . CASP1 - GFP lignin ( fuchsin ) are presented in green magenta respectively . Pictures were obtained from more than 10 roots from each background with similar results . Scale bar = 10 µm .

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(A) CASP1-GFP and lignin deposition in WT, sgn3, rbohD, rbohF and rbohDF. CASP1-GFP and lignin (fuchsin) are presented in green and magenta respectively. Pictures were obtained from more than 10 roots from each background with similar results. Scale bar = 10 µm.


( B ) Time lapse imaging of single - or cotreatment of 10 nM CIF2 with 25 µM cycloheximide ( CHX ) on CASP1 - GFP in cif1 cif2 . Seedlings were pretreated with or without CHX for 30 min transferred onto each medium . Scale bar = 10 µm ( See also movie EV1 ) . ( C ) of ( B ) . Relative numbers of holes in CASP1 - GFP domain after single - or co - treatment with CIF2 or CHX from pictures in ( B ) . Bars are S . D . * indicates statistical significance from other conditions ( p < 0 . 01 ) after one - way ANOVA Tukey . 6 roots in total for each condition were observed during two independent .

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 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 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

(B) Time lapse imaging of single- or cotreatment of 10 nM CIF2 with 25 µM cycloheximide (CHX) on CASP1-GFP in cif1 cif2. Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium. Scale bar = 10 µm (See also movie EV1). (C) Quantification of (B). Relative numbers of holes in CASP1-GFP domain after single- or co-treatment with CIF2 or CHX from the pictures in (B). Bars are S.D. * indicates statistical significance from all other conditions (p<0.01) after one-way ANOVA and Tukey test. 6 roots in total for each condition were observed during two independent tests.


( D ) Fold - change of 930 genes ( p < 0 . 05 log2 ( Fold change ) ≥ 1 or ≤ - 1 at least one time point in one genotype ) after CIF2 treatment at indicated time points in WT , cif1 , 2 sgn3 . Degree of fold changes are shown in color code as indicated .

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(D) Fold-change of 930 genes (p<0.05 and log2(Fold change) ≥1 or ≤ -1 at least one time point in one genotype) after CIF2 treatment at indicated time points in WT, cif1,2 and sgn3. Degree of the fold changes are shown in color code as indicated.