Figure 6 ( B ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( pseudo - colored ) in cortical neurons on the basal condition and on DHPG ( 50μM ) treatment for 5 minutes in the presence or absence of AICAR ( 1mM ) . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm . ( C ) Box plot showing the normalized phospho - eEF2 intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 17 - 46 cells per group from 3 independent platings . ( D ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n = 18 - 57 cells per group from 3 independent platings . ( E ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( Pseudo - colored ) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm . ( F ) Box plot showing phospho - eEF2 intensity distribution across multiple neurons on basal condition ( Vehicle control ) , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 21 - 36 cells per group from 3 independent platings . ( G ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 24 - 52 cells per group from 3 independent platings .

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

Figure 6(B) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (pseudo-colored) in cortical neurons on the basal condition and on DHPG (50μM) treatment for 5 minutes in the presence or absence of AICAR (1mM). MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.(C) Box plot showing the normalized phospho-eEF2 intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=17-46 cells per group from 3 independent platings.(D) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n=18-57 cells per group from 3 independent platings.(E) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (Pseudo-colored) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.(F) Box plot showing phospho-eEF2 intensity distribution across multiple neurons on basal condition (Vehicle control), on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=21-36 cells per group from 3 independent platings.(G) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=24-52 cells per group from 3 independent platings.


Figure 1 ( G ) U2OS ( left panel , n = 6 biological replicates ) or HeLa cells ( right panel , n = 3 biological replicates ) were transfected with the indicated siRNA ; 48 hours post - transfection , they were treated with the radiomimetic antibiotic , phleomycin ( 50 μg / mL ) , and collected at the indicated time points . Flow cytometry analysis of phosphorylated - H2AX signal was used to measure γ - H2AX endogenous signal . Data are represented as a bar graph showing the mean ± SEM , each replicate being represented by a round symbol . Significance was determined by two - way ANOVA followed by a Holm - Sidak ' s test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 0005 . ( H ) U2OS ( n = 8 biological replicates ) were transfected with the indicated siRNA ; 48 hours post - transfection , they were treated with the radiomimetic antibiotic , phleomycin ( 50 μg / mL ) , and collected at the indicated time points . Data are represented as a bar graph showing the mean ± SEM , each replicate being representing as a round symbol . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 .

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

Figure 1(G) U2OS (left panel, n=6 biological replicates) or HeLa cells (right panel, n = 3 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Flow cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal. Data are represented as a bar graph showing the mean ± SEM, each replicate being represented by a round symbol. Significance was determined by two-way ANOVA followed by a Holm-Sidak's test. *P<0.05, **P<0.01, ***P<0.0005.(H) U2OS (n=8 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Data are represented as a bar graph showing the mean ± SEM, each replicate being representing as a round symbol. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.


Figure 2 ( A ) U2OS ( left panel ) , RPE1 - hTERT ( middle panel ) , and HeLa cells ( right panel ) were monitored for their sensitivity to the radiomimetic drug NCS using the SRB assay . For each cell line , the following conditions were used : U2OS cells were transfected with a nontargeting siRNA ( siCtrl ) or an siRNA targeting human POGZ ( siPOGZ - 1 or - 2 ) ; RPE1 - hTERT cells were transduced a control shRNA ( shCtrl ) or a shRNA directed against human POGZ ( shPOGZ - 1 or - 2 ) ; HeLa cells expressing a non - targeting sgRNA ( sgCtrl ) or a sgRNA targeting human POGZ and sub - cloned ( POGZ ∆ - 1 or - 2 ) . Cells were pulsed with NCS at the indicated concentrations for 1 hour , replenished with fresh medium and incubated for 4 days before being processed for SRB assays . Data are represented as a bar graph showing the relative mean ± SEM , each replicate being representing as a round symbol ( 3 biological replicates ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 01 . ( D ) U2OS ( left panel ) and HeLa ( right panel ) cells containing the DR - GFP reporter construct were transfected with the indicated siRNA . 24h post - transfection . Cells were transfected with the I - SceI expression plasmid or an empty vector ( EV ) , and the GFP + population was analyzed 48h post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition provided with I - SceI ( siCtrl , I - SceI ) . Data are represented as the mean ± SEM , each replicate being representing as a round symbol ( n = 3 biological replicates ) . Significance was determined by one - way ANOVA followed by a Dunnett ' s test . * P ≤ 0 . 0001 . ( F ) Quantification of γ - H2AX foci in HeLa cells where POGZ has been targeted by CRISPR technology ( POGZ ∆ - 1 or - 2 ) and in control HeLa cells ( sgCtrl ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered at the indicated time points . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of γ - H2AX foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 0005 . ( G ) Representative images used for quantification in ( F ) . Scale bar = 5 μm .

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

Figure 2(A) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their sensitivity to the radiomimetic drug NCS using the SRB assay. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were pulsed with NCS at the indicated concentrations for 1 hour, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round symbol (3 biological replicates). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.01.(D) U2OS (left panel) and HeLa (right panel) cells containing the DR-GFP reporter construct were transfected with the indicated siRNA. 24h post-transfection. Cells were transfected with the I-SceI expression plasmid or an empty vector (EV), and the GFP+ population was analyzed 48h post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition provided with I-SceI (siCtrl, I-SceI). Data are represented as the mean ± SEM, each replicate being representing as a round symbol (n=3 biological replicates). Significance was determined by one-way ANOVA followed by a Dunnett's test. *P≤0.0001.(F) Quantification of γ-H2AX foci in HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1 or -2) and in control HeLa cells (sgCtrl). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered at the indicated time points. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0005. (G) Representative images used for quantification in (F). Scale bar = 5 μm.


Figure 3 ( A ) U2OS stably expressing mCherry - LacR - FokI were transfected with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield1 and 4 - OHT . Immunofluorescence against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 100 cells per condition were counted ( n = 3 biological replicates ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 005 , * * P ≤ 0 . 0005 . ( B ) U2OS ( left panel ) , RPE1 - hTERT ( middle panel ) , and HeLa cells ( right panel ) were monitored for their capacity to form IR - induced BRCA1 foci . For each cell line , the following conditions were used : U2OS cells were transfected with a nontargeting siRNA ( siCtrl ) or an siRNA targeting human POGZ ( siPOGZ - 1 or - 2 ) ; RPE1 - hTERT cells were transduced a control shRNA ( shCtrl ) or a shRNA directed against human POGZ ( shPOGZ - 1 or - 2 ) ; HeLa cells were expressing a non - targeting sgRNA ( sgCtrl ) or a sgRNA targeting human POGZ and sub - cloned ( POGZ ∆ - 1 or - 2 ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post - exposure to IR . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of ΒRCA1 foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 005 , * * P < 0 . 001 . ( E ) U2OS stably expressing mCherry - LacR - FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield - 1 and 4 - OHT . Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 50 cells per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 0001 . ( G ) U2OS stably expressing mCherry - LacR - FokI were transduced with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield - 1 and 4 - OHT . Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 75 cells per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 005 , * * P < 0 . 0001 .

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

Figure 3(A) U2OS stably expressing mCherry-LacR-FokI were transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield1 and 4-OHT. Immunofluorescence against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 100 cells per condition were counted (n=3 biological replicates). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.005, **P≤0.0005.(B) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their capacity to form IR-induced BRCA1 foci. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells were expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.001.(E) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 50 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.0001.(G) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 75 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001.


Figure 4 ( C ) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR - induced BRCA1 foci . HeLa cells where POGZ has been targeted by CRISPR technology ( POGZ ∆ - 1 ) and in control HeLa cells ( sgCtrl ) were transfected by an empty Flag vector ( EV ) or a sgRNA - resistant FLAG - tagged POGZ cDNA construct corresponding to indicated rescue mutant ( full - length , FL ; POGZ801 - 848 , HPZ ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post - exposure to IR . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of ΒRCA1 foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 0001 . ( D ) Representative images used for quantification in ( C ) . Scale bar = 5 μm . ( E ) Similar as in ( C ) except that γ - H2AX foci were monitored at the indicated time points by confocal microscopy . Data are the total number of γ - H2AX foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 . ( F ) Representative images used for quantification in ( E ) . Scale bar = 5 μm .

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

Figure 4(C) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR-induced BRCA1 foci. HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1) and in control HeLa cells (sgCtrl) were transfected by an empty Flag vector (EV) or a sgRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ801-848, HPZ). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0001. (D) Representative images used for quantification in (C). Scale bar = 5 μm. (E) Similar as in (C) except that γ-H2AX foci were monitored at the indicated time points by confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05. (F) Representative images used for quantification in (E). Scale bar = 5 μm.


Figure 5Observed genotypic distribution of offspring of heterozygous Pogz + / ∆ crosses at specified embryonic day . Each litter is considered a biological replicate . Data are represented as a bar graph showing the mean ± SEM ( n for each embryonic day is specified ) . ( D ) ( E ) The body mass of male wild - type ( WT ) or Pogz + / ∆ mice was monitored weekly for 4 weeks post - weaning . Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) . At least 4 mice per genotype was monitored . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 . ( H ) Quantification of the distance travelled ( left panel ) and the average speed ( right panel ) of each mouse ( n = 6 mice per genotype ) in the open field . Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by unpaired two - tailed t - test . * P < 0 . 005 . ( L ) Plasma was isolated from cardiac punctures of wild - type ( WT ) or Pogz + / ∆ mice ( 8 weeks ) and assessed for circulating levels of specified immunoglobulin isotypes . Each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) ( n = 6 mice per genotype ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 005 .

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

Figure 5Observed genotypic distribution of offspring of heterozygous Pogz+/∆ crosses at specified embryonic day. Each litter is considered a biological replicate. Data are represented as a bar graph showing the mean ± SEM (n for each embryonic day is specified). (D)(E) The body mass of male wild-type (WT) or Pogz+/∆ mice was monitored weekly for 4 weeks post-weaning. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ). At least 4 mice per genotype was monitored. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.(H) Quantification of the distance travelled (left panel) and the average speed (right panel) of each mouse (n=6 mice per genotype) in the open field. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by unpaired two-tailed t-test. *P<0.005.(L) Plasma was isolated from cardiac punctures of wild-type (WT) or Pogz+/∆ mice (8 weeks) and assessed for circulating levels of specified immunoglobulin isotypes. Each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ) (n=6 mice per genotype). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005.


Figure 6 ( A ) Wild - type ( WT ) and Pogz + / ∆ mice were subjected to a lethal dose of ionizing radiation ( 8 . 5 Gy ) before recovering in sterile conditions and being assessed for their sensitivity to IR . Data are represented as a Kaplan - Meier survival curve of each genotype ( ( n = 6 mice per genotype ) . Significance was determined by log - rank ( Mantel - Cox ) test . * P < 0 . 005 . ( D ) MEFs were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay . Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr , replenished with fresh medium and incubated for 4 days before being processed for SRB assays . Data are represented as a bar graph showing the relative mean ± SEM , each replicate being representing as a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 005 , * * P < 0 . 0005 . ( H ) Stimulated CD43 - negative splenocytes were loaded on slides via Cytospin and processed for γ - H2AX immunofluorescence . Cells were fixed , stained , and imaged via confocal microscopy . Data are the percentage of cells in a field of view with indicated γ - H2AX foci and are represented as a bar graph showing the mean ± SEM . At least 100 cells per genotype were counted . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 , * * P < 0 . 005 .

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

Figure 6(A) Wild-type (WT) and Pogz+/∆ mice were subjected to a lethal dose of ionizing radiation (8.5 Gy) before recovering in sterile conditions and being assessed for their sensitivity to IR. Data are represented as a Kaplan-Meier survival curve of each genotype ((n=6 mice per genotype). Significance was determined by log-rank (Mantel-Cox) test. *P<0.005.(D) MEFs were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay. Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005, **P<0.0005.(H) Stimulated CD43-negative splenocytes were loaded on slides via Cytospin and processed for γ-H2AX immunofluorescence. Cells were fixed, stained, and imaged via confocal microscopy. Data are the percentage of cells in a field of view with indicated γ-H2AX foci and are represented as a bar graph showing the mean ± SEM. At least 100 cells per genotype were counted. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05, **P<0.005.


Figure 1B , Representative images ( left panel ) and quantifications ( right panel ) of NNMT immunostaining in tissue sections from primary tumors ( T ) and metastases ( M ) in different breast cancer models , scale bar : 100 µm . n = 5 tissue sections per model . * * P < 0 . 01 ; n . s . , not significant , n . a . , not available ; Mann - Whitney U - test . All data are means ± SD . C , Kaplan - Meier overall survival analysis of breast cancer patients stratified according to NNMT protein levels ( negative or weak , n = 541 ; intermediate , n = 108 ; strong , n = 44 ) in primary ( n = 517 ) and metastatic ( n = 176 ) tumor tissue . Estimated 5 - year overall survival rates for subjects with strong , intermediate , and negative / weak NNMT levels were 43 . 0 ± 10 . 0 % , 69 . 0 ± 6 . 0 % and 73 . 0 ± 2 . 0 % , respectively . * * P < 0 . 01 ; Log - rank test . D , Graphic quantification ( pie charts ) and representative images of immunostaining from 625 primary breast cancer specimens classified according to NNMT protein abundance [ negative ( neg ) / weak , intermediate or strong ] . NNMT levels are significantly higher ( P = 0 , Student ' s T - test ) in ERα negative ( neg ) than in ERα positive ( pos ) breast cancers ( side table ) . Scale bar : 100 µm . E , Kaplan - Meier plot depicting metastasis onset after tumor removal in mice injected orthotopically with SUM159PT WT ( n = 10 ) , KO ( KOd : n = 5 ; KOs : n = 5 ) or KO - NNMT ( KOd - NNMT : n = 5 ; KOs - NNMT : n = 5 ) cells . * P < 0 . 05 ; Log - rank test . F , Bar graph quantification ( left panel ) and representative bioluminescence images ( right panel ) of metastases at day 75 after cancer cell injection of MDA - MB - 231 KO1 - RFP or KO1 - NNMT cells and post - primary tumor removal . n = 7 to 10 animals per group . * P < 0 . 05 ; Mann - Whitney U - test . All data are means ± SD . G , Bar graph quantification ( left panel ) and representative bioluminescence images ( right panel ) of metastases at day 34 after injection of MDA - MB - 231 KO1 - RFP or KO1 - NNMT cells into the tail veins of mice . n = 10 animals per group . * * * P < 0 . 001 ; Mann - Whitney U - test . All data are means ± SD .

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

Figure 1B, Representative images (left panel) and quantifications (right panel) of NNMT immunostaining in tissue sections from primary tumors (T) and metastases (M) in different breast cancer models, scale bar: 100 µm. n = 5 tissue sections per model. **P < 0.01; n.s., not significant, n.a., not available; Mann-Whitney U-test. All data are means ± SD.C, Kaplan-Meier overall survival analysis of breast cancer patients stratified according to NNMT protein levels (negative or weak, n = 541; intermediate, n = 108; strong, n = 44) in primary (n = 517) and metastatic (n =176) tumor tissue. Estimated 5-year overall survival rates for subjects with strong, intermediate, and negative/weak NNMT levels were 43.0 ± 10.0%, 69.0 ± 6.0% and 73.0 ± 2.0%, respectively. **P < 0.01; Log-rank test.D, Graphic quantification (pie charts) and representative images of immunostaining from 625 primary breast cancer specimens classified according to NNMT protein abundance [negative (neg)/weak, intermediate or strong]. NNMT levels are significantly higher (P = 0, Student's T-test) in ERα negative (neg) than in ERα positive (pos) breast cancers (side table). Scale bar: 100 µm.E, Kaplan-Meier plot depicting metastasis onset after tumor removal in mice injected orthotopically with SUM159PT WT (n = 10), KO (KOd: n = 5; KOs: n = 5) or KO-NNMT (KOd-NNMT: n= 5; KOs-NNMT: n= 5) cells. *P < 0.05; Log-rank test.F, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 75 after cancer cell injection of MDA-MB-231 KO1- RFP or KO1- NNMT cells and post-primary tumor removal. n = 7 to 10 animals per group. *P < 0.05; Mann-Whitney U-test. All data are means ± SD. G, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 34 after injection of MDA-MB-231 KO1- RFP or KO1- NNMT cells into the tail veins of mice. n = 10 animals per group. ***P < 0.001; Mann-Whitney U-test. All data are means ± SD.


Figure 2C , Representative histological images of SUM159 PT - WT , - KOd , and KOs tumors immunostained with an anti - human vimentin antibody . n = 5 tumors , 2 sections each . Scale bar : 100 µm . G , Representative images of human COL1A1 immunostaining ( left panel ) and bar graph quantification of COL1A1 positive area ( right panel ) in tissue sections of SUM159PT WT , KOd , and KOs tumors . n = 10 to 15 tumors per model with 2 - 4 tissue sections per tumor . * * P < 0 . 01 ; Kruskal - Wallis test . All data are means ± SD . Scale bar : 200 µm .

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Figure 2C, Representative histological images of SUM159 PT-WT, -KOd, and KOs tumors immunostained with an anti-human vimentin antibody. n = 5 tumors, 2 sections each. Scale bar: 100 µm.G, Representative images of human COL1A1 immunostaining (left panel) and bar graph quantification of COL1A1 positive area (right panel) in tissue sections of SUM159PT WT, KOd, and KOs tumors. n = 10 to 15 tumors per model with 2 - 4 tissue sections per tumor. **P < 0.01; Kruskal-Wallis test. All data are means ± SD. Scale bar: 200 µm.


Figure 3A , Bar graph depicting increased ratio of S - adenosylmethionine ( SAM ) : S - adenosylhomocysteine ( SAH ) in SUM159PT KOd and KOs compared to WT cells . n = 3 technical replicates . * P < 0 . 05 ; * * P < 0 . 01 ; Mann - Whitney U - test . All data are means ± SD . D , Dot plots depicting the overlap of H3K9me3 ChIP - sequencing and mRNA - sequencing data . In the y axis : differential H3K9me3 enrichment at the gene promoter in NNMT KO compared to WT cells . In the x axis : differential mRNA expression of NNMT KO compared to WT cells . n = 3 experimental replicates . Cut - off : adjusted P < 0 . 05 . Bold dots highlight collagen genes and PRDM5 . F , Bar graph representing average PRDM5 mRNA expression in SUM159PT KOs , KOd , and WT cells . n = 3 experimental replicates with 2 technical replicates each . * * * * P < 0 . 0001 ; Mann - Whitney U - test . All data are means ± SD . G , H , Bar graphs representing average collagen gene mRNA expression upon overexpression ( OE ) of PRDM5 in SUM159PT KOd and KOs cells . n = 3 to 5 experimental replicates with 2 technical replicates each . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ; Kruskal - Wallis test . All data are means ± SD .

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

Figure 3A, Bar graph depicting increased ratio of S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) in SUM159PT KOd and KOs compared to WT cells. n = 3 technical replicates. *P < 0.05; **P < 0.01; Mann-Whitney U-test. All data are means ± SD.D, Dot plots depicting the overlap of H3K9me3 ChIP-sequencing and mRNA-sequencing data. In the y axis: differential H3K9me3 enrichment at the gene promoter in NNMT KO compared to WT cells. In the x axis: differential mRNA expression of NNMT KO compared to WT cells. n = 3 experimental replicates. Cut-off: adjusted P < 0.05. Bold dots highlight collagen genes and PRDM5.F, Bar graph representing average PRDM5 mRNA expression in SUM159PT KOs, KOd, and WT cells. n = 3 experimental replicates with 2 technical replicates each. ****P < 0.0001; Mann-Whitney U-test. All data are means ± SD.G, H, Bar graphs representing average collagen gene mRNA expression upon overexpression (OE) of PRDM5 in SUM159PT KOd and KOs cells. n = 3 to 5 experimental replicates with 2 technical replicates each. *P < 0.05, **P < 0.01, n.s., not significant; Kruskal-Wallis test. All data are means ± SD.


Figure 4A , Representative images and bar graph quantification depicting 5 - methylcytosine ( 5 - mC ) signal by immunofluorescence in SUM159PT WT , KOs , and KOd cells . n = 2 experimental replicates with 5 to 6 technical replicates each . * * * P < 0 . 001 ; Kruskal - Wallis test . Data are means ± SD . Scale bar , 50 µm . D , Bar graphs depicting 5 - mC abundance at promoters of the indicated genes shown as fold enrichment of methylated DNA immunoprecipitate ( MedIP ) over IgG control . n = 3 experimental replicates with 2 to 3 technical replicates each . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ; Kruskal - Wallis test . Data are means ± SEM . E , Bar graphs representing average PRDM5 and collagen gene mRNA expression upon 5 - aza treatment in SUM159PT WT , Kos , and KOd cells . n = 3 experimental replicates with 2 technical replicates each . * P < 0 . 05 , n . s . , not significant ; Kruskal - Wallis test . Data are means ± SEM .

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

Figure 4A, Representative images and bar graph quantification depicting 5-methylcytosine (5-mC) signal by immunofluorescence in SUM159PT WT, KOs, and KOd cells. n = 2 experimental replicates with 5 to 6 technical replicates each. ***P < 0.001; Kruskal-Wallis test. Data are means ± SD. Scale bar, 50 µm.D, Bar graphs depicting 5-mC abundance at promoters of the indicated genes shown as fold enrichment of methylated DNA immunoprecipitate (MedIP) over IgG control. n = 3 experimental replicates with 2 to 3 technical replicates each. *P < 0.05, ** P < 0.01, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.E, Bar graphs representing average PRDM5 and collagen gene mRNA expression upon 5- aza treatment in SUM159PT WT, Kos, and KOd cells. n = 3 experimental replicates with 2 technical replicates each. *P < 0.05, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.


Figure 5A , Bar graph quantification ( left panel ) and representative bioluminescence images ( right panel ) of metastases at day 27 after injection of MDA - MB - 231 cells into the tail veins of mice bearing short hairpin sh NT ( non - targeting ) , sh1 or sh2 targeting PRDM5 . Mice were fed with doxycycline food to sustain shRNA expression during the whole experiment . n = 7 to 8 animals per group . * * P < 0 . 01 ; One - way ANOVA . All data are means ± SD . B , Box plots depicting bioluminescence quantification ( left panel ) and representative images ( right panel ) of metastases at day 42 and day 62 after intravenous injection of MDA - MB - 231 KO1 _ RFP , KO1 _ NNMT1 and KO1 _ COL1A1 ( COL1A1 over - expression ) cells . n = 4 to 8 animals per group . * * P < 0 . 01 , * * * P < 0 . 001 ; Day 42 : One - way ANOVA , Day 62 : Student t - test . Boxes define the upper and lower quartiles ; central band indicate the median ; whiskers define max to min values . E , Box plots showing COL1A1 and PRDM5 expression level ( H - Score ) according to the NNMT status ( NNMT negative , n = 17 ; NNMT positive , n = 47 ) within the hormone receptor - negative ( HR - ) tumor samples . * P < 0 . 05 ; Student t - test . Boxes define the upper and lower quartiles ; central band indicate the median ; whiskers define max to min values .

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

Figure 5A, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 27 after injection of MDA-MB-231 cells into the tail veins of mice bearing short hairpin sh NT (non-targeting), sh1 or sh2 targeting PRDM5. Mice were fed with doxycycline food to sustain shRNA expression during the whole experiment. n = 7 to 8 animals per group. **P < 0.01; One-way ANOVA. All data are means ± SD.B, Box plots depicting bioluminescence quantification (left panel) and representative images (right panel) of metastases at day 42 and day 62 after intravenous injection of MDA-MB-231 KO1_RFP, KO1_NNMT1 and KO1_COL1A1 (COL1A1 over-expression) cells. n = 4 to 8 animals per group. **P < 0.01, ***P < 0.001; Day 42: One-way ANOVA, Day 62: Student t-test. Boxes define the upper and lower quartiles; central band indicate the median; whiskers define max to min values.E, Box plots showing COL1A1 and PRDM5 expression level (H-Score) according to the NNMT status (NNMT negative, n = 17; NNMT positive, n = 47) within the hormone receptor-negative (HR-) tumor samples. *P < 0.05; Student t-test. Boxes define the upper and lower quartiles; central band indicate the median; whiskers define max to min values.


Figure 1Western blot of pre - mRNA 3 ' - end processing factors in response to UV treatment ( 40 J / m2 ) of A549 cells ( n = 3 ) , followed by16 hours of recovery . GAPDH was used as a loading control . RT - qPCR assay on nuclear RNA for assessing the uncleaved / total ratio of p53 and TATA - binding protein ( TBP ) pre - mRNAs in A549 cells ( n = 3 ) transfected for 48 hours with two different siRNA targeting PCF11 prior to exposure ( UV + ) or not ( UV - ) to UV irradiation ( 40 J / m2 ) . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m . P values were calculated using two - sided unpaired t - test .

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

Figure 1Western blot of pre-mRNA 3'-end processing factors in response to UV treatment (40 J/m2) of A549 cells (n=3), followed by16 hours of recovery. GAPDH was used as a loading control.RT-qPCR assay on nuclear RNA for assessing the uncleaved/total ratio of p53 and TATA-binding protein (TBP) pre-mRNAs in A549 cells (n=3) transfected for 48 hours with two different siRNA targeting PCF11 prior to exposure (UV+) or not (UV-) to UV irradiation (40 J/m2). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.


Figure 2RT - qPCR analysis on RNA extracted from nucleoplasm and chromatin fractions . The ratio of uncleaved pre - mRNA ( nucleoplasm / chromatin ) was calculated to quantify the level of unprocessed p53 , TBP , WDR13 and GAPDH pre - mRNA ( n = 3 biological replicates ) released in the nucleoplasm compared to the chromatin - bound unprocessed pre - mRNA . WDR13 and GAPDH were included as controls as they have been previously reported to be processed post - and co - transcriptionally , respectively . Data are presented as the mean ± s . e . m . RT - PCR analysis of p53 3 ' flanking region using the same primers employed in the data panel above ( n = 3 ) . Lane 1 is a control PCR amplification of cDNA derived from the reverse transcription of a control mRNA using oligo ( dT ) . Lanes 2 - 7 are PCR amplification of reverse transcribed p53 chromatin - associated pre - mRNA using oligo oligo ( dT ) .

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Figure 2RT-qPCR analysis on RNA extracted from nucleoplasm and chromatin fractions. The ratio of uncleaved pre-mRNA (nucleoplasm/chromatin) was calculated to quantify the level of unprocessed p53, TBP, WDR13 and GAPDH pre-mRNA (n=3 biological replicates) released in the nucleoplasm compared to the chromatin-bound unprocessed pre-mRNA. WDR13 and GAPDH were included as controls as they have been previously reported to be processed post- and co-transcriptionally, respectively. Data are presented as the mean ± s.e.m.RT-PCR analysis of p53 3' flanking region using the same primers employed in the data panel above (n=3). Lane 1 is a control PCR amplification of cDNA derived from the reverse transcription of a control mRNA using oligo (dT). Lanes 2-7 are PCR amplification of reverse transcribed p53 chromatin-associated pre-mRNA using oligo oligo (dT).


Figure 3Representative images of smFISH in untreated ( - UV ) or UV - treated ( + UV ) A549 cells with the indicated probes ( A , B , C and D ) .

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Figure 3Representative images of smFISH in untreated (-UV) or UV-treated (+UV) A549 cells with the indicated probes (A, B, C and D).


Figure 4RT - qPCR assay on nuclear RNA for assessing the uncleaved / total ratio of p53 pre - mRNA in wild type ( WT ) and CoTC deleted ( ΔCoTC ) A549 cells ( n = 3 ) treated with or without UV irradiation ( 40 J / m2 ) . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m . P values were calculated using two - sided unpaired t - test . RT - qPCR measuring relative p53 and p21 mRNA levels in wild type ( WT ) and CoTC - deleted ( ΔCoTC ) cells ( n = 3 ) in response to UV treatment ( 40 J / m2 ) . The expression was normalized to HPRT . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m . P values were calculated using two - sided unpaired t - test . Western blot analysis of p53 and p21 expression in wild type ( WT ) and CoTC deleted ( ΔCoTC ) A549 cells ( n = 3 ) treated with or without UV irradiation ( 40 J / m2 ) .

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Figure 4RT-qPCR assay on nuclear RNA for assessing the uncleaved/total ratio of p53 pre-mRNA in wild type (WT) and CoTC deleted (ΔCoTC) A549 cells (n=3) treated with or without UV irradiation (40 J/m2). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.RT-qPCR measuring relative p53 and p21 mRNA levels in wild type (WT) and CoTC-deleted (ΔCoTC) cells (n=3) in response to UV treatment (40 J/m2). The expression was normalized to HPRT. "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.Western blot analysis of p53 and p21 expression in wild type (WT) and CoTC deleted (ΔCoTC) A549 cells (n=3) treated with or without UV irradiation (40 J/m2).


Figure 6RT - qPCR ( uncleaved / total RNA ) on nuclear RNA extracted from UV - treated or untreated A549 cells ( n = 3 ) , to assess the regulation of 3 ' end processing of 20 pre - mRNAs randomly selected from the previous RNA - sequencing data . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m . RT - qPCR ( Released uncleaved pre - mRNA nucleoplasm / chromatin ) on RNA extracted from the nucleoplasm and the chromatin fractions of A549 UV - treated cells ( n = 3 ) . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m .

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Figure 6RT-qPCR (uncleaved/total RNA) on nuclear RNA extracted from UV-treated or untreated A549 cells (n=3), to assess the regulation of 3'end processing of 20 pre-mRNAs randomly selected from the previous RNA-sequencing data. "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m.RT-qPCR (Released uncleaved pre-mRNA nucleoplasm/chromatin) on RNA extracted from the nucleoplasm and the chromatin fractions of A549 UV-treated cells (n=3). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m.


Figure 1A , B . Height ( A ) and body mass index ( B ) of the participants by age in the diet groups compared to 2 SD intervals ( shaded area ) and median in the Finnish reference population . C . Dietary intake of selected macro - and micronutrients of participants . The shaded areas represent Finnish dietary recommendations for 2 to 5 years olds that covers majority of participants ( THL , 2019 ) . Energy intake recommendations vary by weight and therefore unambiguous limits could not be represented .

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Figure 1A, B. Height (A) and body mass index (B) of the participants by age in the diet groups compared to 2 SD intervals (shaded area) and median in the Finnish reference population.C. Dietary intake of selected macro- and micronutrients of participants. The shaded areas represent Finnish dietary recommendations for 2 to 5 years olds that covers majority of participants (THL, 2019). Energy intake recommendations vary by weight and therefore unambiguous limits could not be represented.


Figure 2A . Serum , plasma or urine levels of vitamin D , vitamin A , folate , zinc , iron and iodine status biomarkers in diet groups . Solid lines indicate cut - offs for deficiency , and dashed lines indicate cut - offs for insufficiency . The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1 . 17 µmol / l and 0 . 83 µmol / l , respectively . Vitamin A status was calculated based on RBP , transthyretin , and CRP . ( Talsma et al , 2015 ) . B . Cholesterol metabolism biomarkers in the diet groups . The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol - related metabolites measured in this study with black font . ( Nelson & Cox , 2012 ; Risley , 2002 ) .

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Figure 2A. Serum, plasma or urine levels of vitamin D, vitamin A, folate, zinc, iron and iodine status biomarkers in diet groups. Solid lines indicate cut-offs for deficiency, and dashed lines indicate cut-offs for insufficiency. The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1.17 µmol/l and 0.83 µmol/l, respectively. Vitamin A status was calculated based on RBP, transthyretin, and CRP.(Talsma et al, 2015).B. Cholesterol metabolism biomarkers in the diet groups. The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol-related metabolites measured in this study with black font. (Nelson & Cox, 2012; Risley, 2002).


Figure 3B . Targeted analysis of bile acid concentrations in serum of omnivores , vegetarians and vegans . C . Amino acid concentrations in untargeted metabolomics . Numeric means and standard deviations in this figure can be found in Appendix Table S4 . # Valine and betaine are two major components under the same m / z - peak , causing uncertainty in the interpretation of valine levels .

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Figure 3B. Targeted analysis of bile acid concentrations in serum of omnivores, vegetarians and vegans.C. Amino acid concentrations in untargeted metabolomics. Numeric means and standard deviations in this figure can be found in Appendix Table S4. #Valine and betaine are two major components under the same m/z-peak, causing uncertainty in the interpretation of valine levels.


Figure 4A . Fatty acid analysis from untargeted MS metabolomics including free alpha - linolenic acid ( ALA ) and docosahexaenoic acid ( DHA ) . B . Carnitine - bound fatty acids commonly found in serum that were detected in mass spectrometry . C . Lysophosphatidylcholine and lysophosphatidylethanolamine with C16 : 0 ( palmitic acid ) and C18 : 1 ( oleic acid ) . D . Combined analysis of all found triglycerides correlating to medium - chain fatty acid lengths and long or very long chain fatty acid lengths .

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Figure 4A. Fatty acid analysis from untargeted MS metabolomics including free alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA).B. Carnitine-bound fatty acids commonly found in serum that were detected in mass spectrometry.C. Lysophosphatidylcholine and lysophosphatidylethanolamine with C16:0 (palmitic acid) and C18:1 (oleic acid).D. Combined analysis of all found triglycerides correlating to medium-chain fatty acid lengths and long or very long chain fatty acid lengths.


Figure 1 ( D , E ) HTM - mediated quantification of MPM - 2 ( D ) and H3S10P ( E ) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 ( USP7i ) for 4 hrs . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm . The quantification is shown to the right of representative images for each analysis . ( * * * p < 0 . 05 ; t - test ) . Grey lines indicate mean values .

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Figure 1(D, E) HTM-mediated quantification of MPM-2 (D) and H3S10P (E) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 (USP7i) for 4 hrs. Nuclei were stained with DAPI (blue). Scale bar, 30 μm. The quantification is shown to the right of representative images for each analysis. (*** p<0.05; t-test). Grey lines indicate mean values.


Figure 2 ( A ) Western Blot showing the levels of USP7 , MPM - 2 , CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2 - 4 hrs ( USP7i ) , an asterisk indicates a non - specific band . ( B ) Immunofluorescence of CCNB1 ( red ) and CCNB1S126P ( green ) in RPE cells in control conditions ( Control ) and after treatment with 25 μM P22077 for 2 hrs ( USP7i ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm .

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Figure 2(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band.(B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.


Figure 3 ( B ) Western blot showing the levels of MPM - 2 , pAKT , AKT , pPRC1 , pKAP , KAP , pMAPKAPK - 2 , pSTAT3 , STAT3 and β - Actin in whole cell extracts of RPE cells treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Ponceau staining is shown as loading control . ( D ) Immunofluorescence of PP2A - A ( green ) in U2OS cells treated with DMSO ( CONTROL ) , 50 μM P22077 ( USP7i ) for 4 hrs or 0 . 5 μM Okadaic acid ( OA ) for 1 hr 30 min . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . ( E ) Western blot showing the levels of MPM - 2 , pAKT , AKT , pPRC1 , pMAPKAPK - 2 , p4e - BP1 , 4e - BP1 and β - Actin , in whole cell extracts of RPE cells treated with DMSO ( Control ) , 25 μM P22077 ( USP7i ) and 10 μM of the PP2A activator DT - 061 for 2 - 4 hrs . Ponceau staining is shown as loading control .

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Figure 3(B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control.(D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.(E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.


Figure 4 ( F ) Western blot showing the levels of MPM - 2 , phosphor - CDK substrate motif [ ( K / H ) pSP ] and β - Actin in whole cell extracts of RPE cells treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid ( OA ) for 2 hrs , 10 nM Calyculin A for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor . Ponceau staining is shown as loading control .

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Figure 4(F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H)pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control.


Figure 5 ( E ) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT - 116 cells treated with DMSO ( Control ) , 50 μM P22077 for 2 - 4 hrs ( USP7i ) , 10 μM RO3306 for 4 hrs ( CDK1i ) or 50 μM P22077 + 10 μM RO3306 for 2 - 4 hrs ( USP7i + CDK1i ) . Ponceau staining is shown as loading control .

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Figure 5(E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.


Figure 6 ( B ) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control , 25 μM P22077 ( USP7i ) , 10 μM RO3306 ( CDK1i ) or 10 μM DT - 061 ( PP2Aa ) , and their combinations for the indicated times . DNA content was followed with Hoechst , and the mitochondrial membrane potential was measured by TMRE . The percentage of apoptotic cells ( TMRE negative ) is indicated for each condition . This experiment was repeated 3 times . ( * p < 0 . 05 , * * p < 0 . 01 , t - test ) . Bars represent mean values ± SEM . ( C ) GLO viability assay in cells treated with 3 independent USP7 inhibitors ( P22077 , BAY 11 - 7082 or HBX 19818 ) alone or in combination with a 24hr treatment of 10 μM RO3306 ( CDK1i ) . This experiment was repeated 3 times . Data represent mean values ± SEM . ( * * * p < 0 . 05 , ANOVA ) .

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Figure 6(B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM.(C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).


f1b , Carboxy - terminal V5immunoblot of full - length ( 68 kDa ) and cleaved ( 36 kDa ) ATG16L1β . Silver stain ( bottom gels ) depicts total protein . Data represent 3 independent experiments . IB , immunoblot . c , TNF - α - mediated processing of humanATG16L1 in monocyte - derived macrophages . Scatter plot of immunoblot densitometry depicts a ratio of cleaved : full - length ATG16L1 ( Extended Data Fig . 2 ) . n = 7 donors . d , TNF - α - mediated processing of murineATG16L1 in macrophages . Immunoblot represents 5 independent experiments . Scatterplot represents data pooled from 5 independent experiments . WT , wild type .

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f1b, Carboxy-terminal V5immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1β. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot.c, TNF-α-mediated processing of humanATG16L1 in monocyte-derived macrophages. Scatter plot of immunoblot densitometry depicts a ratio of cleaved:full-length ATG16L1 (Extended Data Fig. 2). n = 7 donors.d, TNF-α-mediated processing of murineATG16L1 in macrophages. Immunoblot represents 5 independent experiments. Scatterplot represents data pooled from 5 independent experiments. WT, wild type.


f2a , HeLa cells transfected with control siRNA ( siCTL ) or siCaspase 8 were stimulated with 2 . 0 μM staurosporine or 20 ng ml − 1 TNF + 10 μg ml − 1 CHX . b , HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a . c , HeLa cells transfected with control , caspase - 3 - or caspase - 7 - specific siRNAs were stimulated with 20 ng ml − 1 TNF + 10 & amp ; amp ; mgr ; g ml − 1 CHX . d , Wild - type or caspase - 3 - knockout macrophages were stimulated with 2 . 0 µM staurosporine or 20 ng ml − 1 TNF + 10 µg ml − 1 CHX . Data in a - c represent 2 independent experiments ; data in d represent 4 independent experiments .

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f2a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX.b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a.c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &amp;mgr;g ml−1 CHX.d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.


f3a , Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1 . White mask in LC3 channel illustrates punctate LC3 staining , representing LC3 - II . Histogram depicts mean fluorescence intensity of 10 , 000 cells per sample . b , Quantification of mean ATG16L1 fluorescence intensity in a , n = 4 mice . Glu . , glucose . c , Immunoblot of phosphorylated ( p - ) AMPK & amp ; amp ; agr ; ( Thr 172 ) and lipidated LC3 ( LC3 - II ) . d , Quantification of mean LC3 - II area in murine macrophages , n = 4 mice . e , Caspase 3 activity in murine macrophages following glucose starvation , n = 3 mice . RLU , relative light units . f , Quantification of mean LC3 - II area in wild - type ( Casp3 + / + ) or Casp3 - knockout ( Casp3 − / − ) macrophages , n = 3 mice . Data in a - f represent 2 independent experiments .

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f3a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose.c, Immunoblot of phosphorylated (p-) AMPK&amp;agr; (Thr 172) and lipidated LC3 (LC3-II).d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice.e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units.f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a-f represent 2 independent experiments.


f4a , Quantification of ATG16L1 mean fluorescence intensity by image - based flow cytometry of 10 , 000 cells , n = 4 ( wild - type , WT ) , 5 ( T300A ) . Data represent 3 independent experiments . b , Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells , n = 5 . Data represent 2 independent experiments . c , Quantification of mean LC3 - II area of samples in a . Data represent 3 independent experiments . d , Visualization of early autophagosomes by transmission electron micrographs ( TEM ) . Red arrows , early autophagosomes , characterized by vesicular structures with a double - membrane . Blue arrows , multivesicular lysosomal structures , identified by degraded intravesicular cargo . Micrographs representative of 10 - 12 images per donor , n = 4 ( WT ) , n = 3 ( T300A ) . A total of 29 ( WT ) and 26 ( T300A ) images containing autophagosomes was analysed . Histogram quantifies autophagic vesicles as a ratio of autophagosome : cytosolic area .

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f4a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments.d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10-12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.


f5a - c , Y . enterocolitica colony forming units ( c . f . u . ) in human and murine macrophages infected for 6 h ( 20 m . o . i . ) . Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype , total n = 16 ( non - risk , WT ) , 15 ( T300A , risk ) . Data in b represent 3 independent experiments , n = 4 mice . Data in c represent 3 independent experiments , n = 6 . d , TNF - α , IL - 1β and IL - 6 levels in culture media of WT and T316A macrophages infected with 20 m . o . i . Y . enterocolitica . Data represent 2 independent experiments , n = 4 mice . e , Ileum and colon histology ( haematoxylin and eosin staining ) of WT mice . Images are representative of 3 ( PBS ) and 8 ( Y . enterocolitica ) treated male mice . f , Measurement of cytokine mRNA transcripts in mesenteric lymph nodes . Data are pooled from 2 independent experiments of 3 mice ( PBS ) and 7 mice ( Y . enterocolitica ) per experiment . Transcripts are normalized to Gapdh ( 2 − Dgr ; Ct ) . g , Y . enterocolitica c . f . u . in mesenteric lymph nodes of WT and T316A knock - in mice 48 h following oral gavage . Data represent 3 independent experiments , n = 9 male mice .

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f5a-c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6.d, TNF-α, IL-1β and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice.e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice.f, Measurement of cytokine mRNA transcripts in mesenteric lymph nodes. Data are pooled from 2 independent experiments of 3 mice (PBS) and 7 mice (Y. enterocolitica) per experiment. Transcripts are normalized to Gapdh (2−Dgr;Ct).g, Y. enterocolitica c.f.u. in mesenteric lymph nodes of WT and T316A knock-in mice 48 h following oral gavage. Data represent 3 independent experiments, n = 9 male mice.


Figure 1A . ATPase activity of ABCC3 wild - type ( WT ) and E1451Q in nanodiscs . The data points for WT and E1451Q are fitted with a Michaelis - Menten equation . All data points are the average of three independent experiments in nanodiscs , and error bars represent standard deviation . B . Substrate - stimulated ATPase activities of ABCC3 WT and E1451Q upon addition of substrate E217βG or DHEAS . All data points are the average of three independent experiments in nanodiscs , and error bars represent standard deviation .

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Figure 1A. ATPase activity of ABCC3 wild-type (WT) and E1451Q in nanodiscs. The data points for WT and E1451Q are fitted with a Michaelis-Menten equation. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.B. Substrate-stimulated ATPase activities of ABCC3 WT and E1451Q upon addition of substrate E217βG or DHEAS. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.


Figure 2C . Relative ATPase activities of ABCC3 WT and mutants in the presence of 2 mM ATP upon addition of E217βG . The relative activity represents the substrate - stimulated activity of ABCC3 WT or its mutants harboring mutation of residues at the substrate - binding pocket . For ( C ) the activities of ABCC3 WT are set as 100 % . One - way analysis of variance ( ANOVA ) is used for the comparison of statistical significance of mutants and WT . The P values of < 0 . 05 , 0 . 01 , and 0 . 001 are indicated with * , * * and * * * , respectively . D . Transport [ 3H ] - E217βG activity assays of ABCC3 WT and mutants . For ( D ) , the activities of ABCC3 WT are set as 100 % . One - way analysis of variance ( ANOVA ) is used for the comparison of statistical significance of mutants and WT . The P values of < 0 . 05 , 0 . 01 , and 0 . 001 are indicated with * , * * and * * * , respectively .

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Figure 2C. Relative ATPase activities of ABCC3 WT and mutants in the presence of 2 mM ATP upon addition of E217βG. The relative activity represents the substrate-stimulated activity of ABCC3 WT or its mutants harboring mutation of residues at the substrate-binding pocket. For (C) the activities of ABCC3 WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.D. Transport [3H]-E217βG activity assays of ABCC3 WT and mutants. For (D), the activities of ABCC3 WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.


Figure 3C . Relative ATPase activities of ABCC3 WT and mutants in nanodiscs upon addition of DHEAS and 2 mM ATP . The relative activity represents the substrate - stimulated activity of ABCC3 WT or mutants harboring mutation of residues at the substrate - binding pocket . E . Relative ATPase activities of ABCC3 WT and mutants K318A , T535A upon addition of E217βG . For ( C ) and ( E ) , the activities of ABCC3 WT are set as 100 % . One - way ANOVA is used for the comparison of statistical significance of mutants and WT . The P values of < 0 . 05 , 0 . 01 , and 0 . 001 are indicated with * , * * and * * * , respectively .

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Figure 3C. Relative ATPase activities of ABCC3 WT and mutants in nanodiscs upon addition of DHEAS and 2 mM ATP. The relative activity represents the substrate-stimulated activity of ABCC3 WT or mutants harboring mutation of residues at the substrate-binding pocket.E. Relative ATPase activities of ABCC3 WT and mutants K318A, T535A upon addition of E217βG. For (C) and (E), the activities of ABCC3 WT are set as 100%. One-way ANOVA is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.


Figure 1A Representative picture of plants carrying pASDOG1 : : LUC sprayed with mock solution , ABA or GA and imaged 24 h later . The full pictures are shown in Fig EV2 and Appendix Fig S1 . Scale bar , 1 cm . B Quantification of emitted light intensity per plant after treatment with ABA and GA , compared with mock treatment . C Antisense expression is reduced in response to drought conditions . The full pictures are shown in Fig EV3 . Scale bar , 1 cm . D Quantification of emitted light intensity per plant for control and drought - treated plants . E Strand - specific RT - qPCR asDOG1 expression analysis of 3 - day Col - 0 ( WT ) drought - treated vs . control plants . F The asDOG1 expression level ( black line ) is reduced while DOG1 sense expression ( grey line ) is increased after application of ABA . Leaves of mature Col - 0 ( WT ) 40 - day - old plants were sprayed with ABA and collected 0 , 6 and 10 h later for RNA extraction and RT - qPCR . Signals were normalized against the level of the UBC transcript . The data points are the averages for at least three biological replicates and are normalized against the mock - treated value . Error bars represent standard deviation . * and * * * represent t - test P - values of < 0 . 05 and < 0 . 001 , respectively .

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Figure 1A Representative picture of plants carrying pASDOG1::LUC sprayed with mock solution, ABA or GA and imaged 24 h later. The full pictures are shown in Fig EV2 and Appendix Fig S1. Scale bar, 1 cm.B Quantification of emitted light intensity per plant after treatment with ABA and GA, compared with mock treatment.C Antisense expression is reduced in response to drought conditions. The full pictures are shown in Fig EV3. Scale bar, 1 cm.D Quantification of emitted light intensity per plant for control and drought-treated plants.E Strand-specific RT-qPCR asDOG1 expression analysis of 3-day Col-0 (WT) drought-treated vs. control plants.F The asDOG1 expression level (black line) is reduced while DOG1 sense expression (grey line) is increased after application of ABA. Leaves of mature Col-0 (WT) 40-day-old plants were sprayed with ABA and collected 0, 6 and 10 h later for RNA extraction and RT-qPCR. Signals were normalized against the level of the UBC transcript. The data points are the averages for at least three biological replicates and are normalized against the mock-treated value. Error bars represent standard deviation.* and *** represent t-test P-values of < 0.05 and < 0.001, respectively.


Figure 2A Col - 0 ( WT ) and dog1 - 3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days . A picture of a representative experiment is shown . Scale bar , 2 cm . B Quantification of recovered and dead Col - 0 ( WT ) , null - 3 and dog1 - 4 plants ; plants were scored as dead when unable to grow and produced offspring after an extended recovery period . C Analysis of DOG1 sense expression in drought - treated vs . control Col - 0 ( WT ) and dog1 - 3 mutant plants . D dog1 - 3 mutant plants are defective in the regulation of the majority of the tested drought marker genes . Col - 0 ( WT ) and dog1 - 3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT - qPCR . Error bars represent standard deviation , and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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Figure 2A Col-0 (WT) and dog1-3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days. A picture of a representative experiment is shown. Scale bar, 2 cm.B Quantification of recovered and dead Col-0 (WT), null-3 and dog1-4 plants; plants were scored as dead when unable to grow and produced offspring after an extended recovery period.C Analysis of DOG1 sense expression in drought-treated vs. control Col-0 (WT) and dog1-3 mutant plants.D dog1-3 mutant plants are defective in the regulation of the majority of the tested drought marker genes. Col-0 (WT) and dog1-3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT-qPCR. Error bars represent standard deviation, and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


Figure 3A Plants expressing LUC fused with full - length DOG1 ( pDOG1LUC : : DOG1 ) were sprayed with mock solution or ABA 10 , 20 and 40 days after germination and analysed 24 h after treatment . The graphs show mean emitted light intensity per plant . B Representative picture of mock - and ABA - treated pDOG1LUC : : DOG1 plants . The full picture is shown in Fig EV2C and D . Scale bar , 1 cm . C A truncated construct that lacks the DOG1 antisense promoter region ( pDOG1shDOG1 : : LUC ) is not induced by ABA and is highly expressed throughout development . The graphs show mean emitted light intensity per plant . D Representative picture of mock - and ABA - treated pDOG1shDOG1 : : LUC plants . Scale bar : 1cm . E RT - qPCR analysis of DOG1 sense mRNA level . F Strand - specific RT - qPCR analysis of antisense levels after 10 h of ABA treatment in 10 - , 20 - and 40 - day - old Col - 0 plants . G The pASDOG1 : : LUC reporter is silenced by the application of ABA , while mutation of TATA elements in the asDOG1 promoter ( pASDOG1 ∆ TATA : : LUC ) leads to attenuation of expression in the presence and absence of ABA . H Mutation of TATA elements in the asDOG1 promoter leads to high - level DOG1 sense expression and non - responsiveness to ABA . Error bars represent standard deviation and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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Figure 3A Plants expressing LUC fused with full-length DOG1 (pDOG1LUC::DOG1) were sprayed with mock solution or ABA 10, 20 and 40 days after germination and analysed 24 h after treatment. The graphs show mean emitted light intensity per plant.B Representative picture of mock- and ABA-treated pDOG1LUC::DOG1 plants. The full picture is shown in Fig EV2C and D. Scale bar, 1 cm.C A truncated construct that lacks the DOG1 antisense promoter region (pDOG1shDOG1::LUC) is not induced by ABA and is highly expressed throughout development. The graphs show mean emitted light intensity per plant.D Representative picture of mock- and ABA-treated pDOG1shDOG1::LUC plants. Scale bar: 1cm.E RT-qPCR analysis of DOG1 sense mRNA level.F Strand-specific RT-qPCR analysis of antisense levels after 10 h of ABA treatment in 10-, 20- and 40-day-old Col-0 plants.G The pASDOG1::LUC reporter is silenced by the application of ABA, while mutation of TATA elements in the asDOG1 promoter (pASDOG1∆TATA::LUC) leads to attenuation of expression in the presence and absence of ABA.H Mutation of TATA elements in the asDOG1 promoter leads to high-level DOG1 sense expression and non-responsiveness to ABA. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


Figure 4A Plants expressing the truncated asDOG1 promoter - deficient version pDOG1shDOG1 : : LUC are more resistant to drought than plants expressing the full - length genomic version pDOG1 : : LUC - DOG1 . Three independent lines are shown for each construct , following 10 days of water deprivation . Scale bar , 2 cm . B RT - qPCR quantification of selected drought marker genes in mock - and ABA - treated Col - 0 ( WT ) and two independent pDOG1shDOG1 : : LUC transgenic lines . Error bars represent standard deviation and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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Figure 4A Plants expressing the truncated asDOG1 promoter-deficient version pDOG1shDOG1::LUC are more resistant to drought than plants expressing the full-length genomic version pDOG1::LUC-DOG1. Three independent lines are shown for each construct, following 10 days of water deprivation. Scale bar, 2 cm.B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


Figure 1 ( D , E ) Representative immunofluorescence microscopy images of the distribution of septin 7 ( red ) or borg3 or borg4 ( green ) in young , aged and aged LT - HSCs treated with CASIN ( 5µM ) . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm .

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Figure 1(D,E) Representative immunofluorescence microscopy images of the distribution of septin 7 (red) or borg3 or borg4 (green) in young, aged and aged LT-HSCs treated with CASIN (5µM). Nuclei were stained with DAPI (blue), scale bar= 5µm.