Figure 6A . Survival status of WT and Trim50 - / - mice after intraperitoneal injection with LPS ( n = 10 mice / group ) . Data information : P - values are shown , two - way ANOVA ( A ) Data are representative of three biological replicates with similar results . ( mean ± SDB . ELISA analysis of IL - 1β , TNF - α and IL - 6 in serum of WT or Trim50 - / - mice after i . p . injection with LPS for 6h ( n = 4 - 6 mice / group ) . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDC . Histopathological analysis of the lung tissues by hematoxylin and eosin ( H & E ) staining . The quantified lung injury was depicted by defined clinical parameters in ALI score ( n = 6 mice / group ) . Scale bars : 50μm . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDD . Flow cytometry analysis of the neutrophils ( left ) and Ly6C + monocytes ( right ) in peritoneum of WT or Trim50 - / - mice after i . p . injection with Alum . ( n = 6 mice / group ) . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDE . WT or Trim50 - / - mice were i . p . injected with FA for 36h , followed with immunoblot analysis of NLRP3 , pro - IL - 1β , Caspase1 p20 , IL - 1β in the kidney tissue samples . Data information : P - values are shown , two - way ANOVA ( A ) or two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDF . H & E staining of kidney tissue sections . Scale bars : 20μm . G . Periodic - acid - Schiff ( PAS ) staining of kidney tissue sections . Scale bars : 20μm .

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

Figure 6A. Survival status of WT and Trim50-/- mice after intraperitoneal injection with LPS (n=10 mice/group). Data information: P-values are shown, two-way ANOVA (A) Data are representative of three biological replicates with similar results. (mean ± SDB. ELISA analysis of IL-1β, TNF-α and IL-6 in serum of WT or Trim50-/- mice after i.p. injection with LPS for 6h (n=4-6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDC. Histopathological analysis of the lung tissues by hematoxylin and eosin (H&E) staining. The quantified lung injury was depicted by defined clinical parameters in ALI score (n=6 mice/group). Scale bars: 50μm. Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDD. Flow cytometry analysis of the neutrophils (left) and Ly6C+ monocytes (right) in peritoneum of WT or Trim50-/- mice after i.p. injection with Alum. (n=6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDE. WT or Trim50-/- mice were i.p. injected with FA for 36h, followed with immunoblot analysis of NLRP3, pro-IL-1β, Caspase1 p20, IL-1β in the kidney tissue samples. Data information: P-values are shown, two-way ANOVA (A) or two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDF. H&E staining of kidney tissue sections. Scale bars: 20μm.G. Periodic-acid-Schiff (PAS) staining of kidney tissue sections. Scale bars: 20μm.


Figure 1Representative time - lapse image series of exponentially growing M . smegmatis P2P1recA - GFPwt ( C ) and P2P1recA - GFPdes ( D ) reporters . Phase - contrast and green fluorescence are merged . Numbers represent hours . Scale bar , 10 µm . Histograms showing the distributions of single - cell fluorescence ( n = 204 ) averaged over the lifetime of the cell ( right panels ) . Black line indicates fitting of the data with a Lognormal function ( C ) or with the sum of three Gaussians ( D ) . Insets : P2P1recA - GFPwt population ' s geometric mean ( Gµ ) calculated by computing the logarithm of the single - cell fluorescence values , the mean of the logarithms and its antilog , P2P1recA - GFPdes subpopulations ' means ( µ ) , and R2 values . The two distributions show that the destabilized GFPdes is more suitable to monitor the dynamics of recA expression and to detect subpopulations , which are masked by using the stable GFPwt variant . E Spearman ' s correlation between single - cell P2P1recA - GFPdes fluorescence range and VMR of fluorescence over the lifetime of the cell ( n = 204 ) . The data shown are from 3 independent experiments . Dashed lines separate different levels of fluorescence dispersion over the lifetimes of single - cells . Colors denote cells belonging to underdispersed ( gray ) , dispersed ( dark green ) , and highly dispersed ( light green ) subpopulations . F P2P1recA - GFPdes fluorescence range over the lifetime of the cell of subpopulations segregated by fluorescence VMR ( 40 < n < 112 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significant difference by Kruskal - Wallis and Dunn ' s multiple comparison test : * * * * p < 0 . 0001 . G Single - cell time traces of P2P1recA - GFPdes fluorescence expressed as a percent of generation time of subpopulations segregated by fluorescence VMR . The average fluorescence ( solid black lines ) and half of the generation time ( dashed lines ) are indicated . Population statistics ( n = 204 ) : 38 % ( 0 < VMR ≤ 1 ) ; 44 % ( 1 < VMR ≤ 10 ) ; 18 % ( VMR > 10 ) . The data shown are from 3 independent experiments . H Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time . Dark green bars indicate moderately pulsing cells ( n = 165 ) . Light green bars indicate highly pulsing cells ( n = 66 ) . Significance by Mann - Whitney test . The data shown are from 3 independent experiments . Peaks mainly occur between 40 % and 70 % of the generation time .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Representative time-lapse image series of exponentially growing M. smegmatis P2P1recA-GFPwt (C) and P2P1recA-GFPdes (D) reporters. Phase-contrast and green fluorescence are merged. Numbers represent hours. Scale bar, 10 µm. Histograms showing the distributions of single-cell fluorescence (n = 204) averaged over the lifetime of the cell (right panels). Black line indicates fitting of the data with a Lognormal function (C) or with the sum of three Gaussians (D). Insets: P2P1recA-GFPwt population's geometric mean (Gµ) calculated by computing the logarithm of the single-cell fluorescence values, the mean of the logarithms and its antilog, P2P1recA-GFPdes subpopulations' means (µ), and R2 values. The two distributions show that the destabilized GFPdes is more suitable to monitor the dynamics of recA expression and to detect subpopulations, which are masked by using the stable GFPwt variant. E Spearman's correlation between single-cell P2P1recA-GFPdes fluorescence range and VMR of fluorescence over the lifetime of the cell (n = 204). The data shown are from 3 independent experiments. Dashed lines separate different levels of fluorescence dispersion over the lifetimes of single-cells. Colors denote cells belonging to underdispersed (gray), dispersed (dark green), and highly dispersed (light green) subpopulations. F P2P1recA-GFPdes fluorescence range over the lifetime of the cell of subpopulations segregated by fluorescence VMR (40 < n < 112). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001. G Single-cell time traces of P2P1recA-GFPdes fluorescence expressed as a percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 204): 38% (0<VMR≤1); 44% (1<VMR≤10); 18% (VMR>10). The data shown are from 3 independent experiments. H Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Dark green bars indicate moderately pulsing cells (n = 165). Light green bars indicate highly pulsing cells (n = 66). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 40% and 70% of the generation time.


Figure 2Color - coded single - cell time traces of the four reporters expressed as percent of the generation time ( B ) . Population coefficients of variation of fluorescence ( % ) are in color - coded boxes . Single - cell VMR of fluorescence ( C ) and fluorescence averaged over the lifetime of the cell ( D ) in exponential phase . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments ( n = 105 cells per strain ) . Single - cell fluorescence decreases but variation does not change in strains devoid of positive regulation . Single - cell fluorescence increases and variation decreases in the strain devoid of negative regulation . Color - coded time - traces of fluorescence ( left panel ) averaged for 50 individual cells per strain ( mean ± SD ) treated with MMC ( 0 . 5 μg / ml ) for 2 hours ( gray shading ) . Fluorescence induction during 6 hours from MMC exposure ( right panel ) . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments . Single - cell analysis of independent lineages separated by black vertical lines . Color - coded traces represent single - cell fluorescence of random descendants monitored in fresh 7H9 medium for 24 hours ( n = 20 ) . White and gray vertical bands represents cell divisions . The data shown are from 2 independent experiments . Aligned pulse profiles averaged for each reporter strain ( color - coded as in A ) . Error bars are SD ( 4 < n < 37 ) . The data shown are from 2 independent experiments . Pulses are abrogated in the strain devoid of negative regulation . Analysis of pulse characteristics . Black lines indicate means and SD ( 52 < n < 88 ) . Significance by Kruskal - Wallis nd Dunn ' s multiple comparison test : ns , not significant ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments . Histogram showing the distribution of the fluorescence peak time of individual cells expressed as a percent of the generation time ( 52 < n < 88 ) . Different strains are color - coded . Statistical analysis by one - way ANOVA and Holm - Sidak ' s multiple comparisons test . Pulses are weaker and fewer in the strain devoid of positive regulation but are not displaced relative to the generation time .

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 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 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 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 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 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

Figure 2Color-coded single-cell time traces of the four reporters expressed as percent of the generation time (B). Population coefficients of variation of fluorescence (%) are in color-coded boxes. Single-cell VMR of fluorescence (C) and fluorescence averaged over the lifetime of the cell (D) in exponential phase. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ***p = 0.0002; ****p < 0.0001. The data shown are from 2 independent experiments (n = 105 cells per strain). Single-cell fluorescence decreases but variation does not change in strains devoid of positive regulation. Single-cell fluorescence increases and variation decreases in the strain devoid of negative regulation.Color-coded time-traces of fluorescence (left panel) averaged for 50 individual cells per strain (mean ± SD) treated with MMC (0.5 μg/ml) for 2 hours (gray shading). Fluorescence induction during 6 hours from MMC exposure (right panel). Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.Single-cell analysis of independent lineages separated by black vertical lines. Color-coded traces represent single-cell fluorescence of random descendants monitored in fresh 7H9 medium for 24 hours (n = 20). White and gray vertical bands represents cell divisions. The data shown are from 2 independent experiments.Aligned pulse profiles averaged for each reporter strain (color-coded as in A). Error bars are SD (4 < n < 37). The data shown are from 2 independent experiments. Pulses are abrogated in the strain devoid of negative regulation.Analysis of pulse characteristics. Black lines indicate means and SD (52 < n < 88). Significance by Kruskal-Wallis nd Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.Histogram showing the distribution of the fluorescence peak time of individual cells expressed as a percent of the generation time (52 < n < 88). Different strains are color-coded. Statistical analysis by one-way ANOVA and Holm-Sidak's multiple comparisons test. Pulses are weaker and fewer in the strain devoid of positive regulation but are not displaced relative to the generation time.


Figure 3Spearman ' s correlation between green ( A - D ) or yellow ( E ) fluorescence range and red fluorescence range over the lifetime of the cell ( 105 < n < 153 ) . The data shown are from 2 independent experiments . On the x - axes P2P1recA - GFPdes ( A - D ) or a C - term translational reporter of SSBa fused to mCitrinewt ( E ) . On the y - axes , a C - term translational reporter of RecA fused to mCherrywt ( A and E ) , transcriptional reporters of ruvC ( B ) and radA ( C ) , and mCherrydes expressed from a constitutive control promoter ( D ) . Colors ( A - D ) indicate subpopulations segregated by VMR of P2P1recA - GFPdes : gray ( 0 < VMR ≤ 1 ) , dark green ( 1 < VMR ≤ 10 ) , and light green ( VMR > 10 ) . Colors ( E ) indicate subpopulations segregated by VMR of RecA - mCherrywt : gray ( 0 < VMR ≤ 1 ) , plum ( 1 < VMR ≤ 10 ) , and magenta ( VMR > 10 ) . Pearson ' s correlation between green ( F - I ) or yellow ( J ) fluorescence and red fluorescence , measured 3 hours after MMC exposure ( 203 < n < 314 ) . The data shown are from 2 independent experiments . On the x - axes P2P1recA - GFPdes ( F - I ) or a C - term translational reporter of SSBa fused to mCitrinewt ( J ) . On the y - axes , a C - term translational reporter of RecA fused to mCherrywt ( F and J ) , transcriptional reporters of ruvC ( G ) and radA ( H ) , and mCherrydes expressed from a constitutive control promoter ( I ) . Positive correlations between recA and other DNA - damage markers both under normal growth conditions , where spontaneous DNA - damage events occur at the subpopulation level , and in the presence of an alkylating agent that induces double - strand breaks in the entire population , validate recA as a reliable proxy for DNA damage in single cells .

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 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

Figure 3Spearman's correlation between green (A-D) or yellow (E) fluorescence range and red fluorescence range over the lifetime of the cell (105 < n < 153). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (A-D) or a C-term translational reporter of SSBa fused to mCitrinewt (E). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (A and E), transcriptional reporters of ruvC (B) and radA (C), and mCherrydes expressed from a constitutive control promoter (D). Colors (A-D) indicate subpopulations segregated by VMR of P2P1recA-GFPdes: gray (0<VMR≤1), dark green (1<VMR≤10), and light green (VMR>10). Colors (E) indicate subpopulations segregated by VMR of RecA-mCherrywt: gray (0<VMR≤1), plum (1<VMR≤10), and magenta (VMR>10).Pearson's correlation between green (F-I) or yellow (J) fluorescence and red fluorescence, measured 3 hours after MMC exposure (203 < n < 314). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (F-I) or a C-term translational reporter of SSBa fused to mCitrinewt (J). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (F and J), transcriptional reporters of ruvC (G) and radA (H), and mCherrydes expressed from a constitutive control promoter (I). Positive correlations between recA and other DNA-damage markers both under normal growth conditions, where spontaneous DNA-damage events occur at the subpopulation level, and in the presence of an alkylating agent that induces double-strand breaks in the entire population, validate recA as a reliable proxy for DNA damage in single cells.


Figure 4Representative time - lapse image series of the merodiploid recA reporter strain ( P2P1recA - GFPdes _ RecA - mCherrywt ) cultured in fresh ( A ) or spent ( B ) flow medium ( see Materials and Methods ) . Fresh 7H9 medium is unlabeled ; spent medium ( S ) , ciprofloxacin 1 µg / ml in fresh ( CIP ) or spent ( S - CIP ) medium , and SYTOX 0 . 5 µM ( SX ) are labeled . Phase - contrast ( gray ) , GFPdes ( green ) , and mCherrywt ( magenta ) fluorescence are merged . Fluorescence images of each channel are scaled to the brightest frame . Light - green background in the first 4 frames ( B ) is due to the autofluorescence of spent medium . Cells were imaged at 30 - min intervals and numbers represent hours . Scale bar , 10 µm . Representative time traces of individual lineages of cells fed with fresh medium ( C ) or spent medium ( D ) . Different styles are used for lineages with different fates : death and lysis ( L ) ; SYTOX - positive ( SX + ) ; and survival ( S ) . Line breaks represent division events in the same lineage , where a single random daughter cell is tracked for clarity . Vertical bars represent fresh ( white ) or spent ( dark shading ) 7H9 medium , CIP ( yellow ) , and SX ( blue ) . Comparison of single - cell growth rates prior to CIP exposure for " D " cells that die and " S " cells that survive and regrow after CIP washout ( E ) , and lag time of regrowth ( F ) . Cells were cultured in fresh ( EXP ) or spent ( STAT ) flow medium . Gray and black lines indicate mean ± SD . Significance by Kruskal - Wallis and Dunn ' s multiple comparison test ( E ) and unpaired Mann - Whitney test ( F ) : ns , not significant ; * * * * p < 0 . 0001 ; 6 < n < 84 . The data shown are from 2 independent experiments . Single - cell fate is independent of the growth rate before exposure to the drug . However , cells cultured in spent medium ( STAT ) grow at significantly slower rates , survive at higher frequencies , and recover faster after CIP washout compared to cells cultured in fresh medium ( EXP ) . Representative GFPdes ( G ) and mCherrywt ( H ) fluorescence time - traces for single - cell lineages of cells cultured in fresh medium ( EXP ) and cells cultured in spent medium ( STAT ) , and having different fates . Vertical bars are color - coded as in C and D . Single - cell fate as a function of VMR of P2P1recA - GFPdes ( I ) and RecA - mCherrywt ( J ) fluorescence prior to CIP exposure . Significance by Chi - square test of independence for cells that die ( n = 84 ) and cells that survive ( n = 54 ) . The data shown are from 2 independent experiments . Cells that do not experience DNA damage prior to drug exposure ( RecA - non - pulsing cells ) survive more than cells that do experience DNA damage ( RecA - pulsing cells ) .

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

Figure 4Representative time-lapse image series of the merodiploid recA reporter strain (P2P1recA-GFPdes_RecA-mCherrywt) cultured in fresh (A) or spent (B) flow medium (see Materials and Methods). Fresh 7H9 medium is unlabeled; spent medium (S), ciprofloxacin 1 µg/ml in fresh (CIP) or spent (S-CIP) medium, and SYTOX 0.5 µM (SX) are labeled. Phase-contrast (gray), GFPdes (green), and mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Light-green background in the first 4 frames (B) is due to the autofluorescence of spent medium. Cells were imaged at 30-min intervals and numbers represent hours. Scale bar, 10 µm.Representative time traces of individual lineages of cells fed with fresh medium (C) or spent medium (D). Different styles are used for lineages with different fates: death and lysis (L); SYTOX-positive (SX+); and survival (S). Line breaks represent division events in the same lineage, where a single random daughter cell is tracked for clarity. Vertical bars represent fresh (white) or spent (dark shading) 7H9 medium, CIP (yellow), and SX (blue).Comparison of single-cell growth rates prior to CIP exposure for "D" cells that die and "S" cells that survive and regrow after CIP washout (E), and lag time of regrowth (F). Cells were cultured in fresh (EXP) or spent (STAT) flow medium. Gray and black lines indicate mean ± SD. Significance by Kruskal-Wallis and Dunn's multiple comparison test (E) and unpaired Mann-Whitney test (F): ns, not significant; ****p < 0.0001; 6 < n < 84. The data shown are from 2 independent experiments. Single-cell fate is independent of the growth rate before exposure to the drug. However, cells cultured in spent medium (STAT) grow at significantly slower rates, survive at higher frequencies, and recover faster after CIP washout compared to cells cultured in fresh medium (EXP).Representative GFPdes (G) and mCherrywt (H) fluorescence time-traces for single-cell lineages of cells cultured in fresh medium (EXP) and cells cultured in spent medium (STAT), and having different fates. Vertical bars are color-coded as in C and D.Single-cell fate as a function of VMR of P2P1recA-GFPdes (I) and RecA-mCherrywt (J) fluorescence prior to CIP exposure. Significance by Chi-square test of independence for cells that die (n = 84) and cells that survive (n = 54). The data shown are from 2 independent experiments. Cells that do not experience DNA damage prior to drug exposure (RecA-non-pulsing cells) survive more than cells that do experience DNA damage (RecA-pulsing cells).


Figure 5Spearman ' s correlation between single - cell RecA - mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell ( n = 360 ) . The data shown are from 3 independent experiments . Dashed lines separate different levels of dispersion , and colors denote subpopulations that are underdispersed ( gray ) , dispersed ( plum ) , or highly dispersed ( magenta ) . RecA - mCherrywt fluorescence of subpopulations segregated by fluorescence VMR ( 58 < n < 159 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significant difference by Kruskal - Wallis and Dunn ' s multiple comparison test : * * * * p < 0 . 0001 . Single - cell time traces of RecA - mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR . The average fluorescence ( solid black lines ) and half of the generation time ( dashed lines ) are indicated . Population statistics ( n = 360 ) : 40 % ( 0 < VMR ≤ 1 ) ; 44 % ( 1 < VMR ≤ 10 ) ; 16 % ( VMR > 10 ) . The data shown are from 3 independent experiments . Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time . Bars shaded in plum indicate moderately pulsing cells ( n = 185 ) and magenta bars indicate highly pulsing cells ( n = 57 ) . Significance by Mann - Whitney test . The data shown are from 3 independent experiments . Peaks mainly occur between 80 % and 100 % of the generation time . Single - cell size ( E ) and growth rate ( F ) averaged over the lifetime of the cell ( 58 < n < 159 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * p = 0 . 002 ; * * * * p < 0 . 0001 . Cells experiencing more DNA damage ( VMR > 10 ) are slightly larger and grow at slower rates .

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Figure 5Spearman's correlation between single-cell RecA-mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell (n = 360). The data shown are from 3 independent experiments. Dashed lines separate different levels of dispersion, and colors denote subpopulations that are underdispersed (gray), dispersed (plum), or highly dispersed (magenta). RecA-mCherrywt fluorescence of subpopulations segregated by fluorescence VMR (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001.Single-cell time traces of RecA-mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 360): 40% (0<VMR≤1); 44% (1<VMR≤10); 16% (VMR>10). The data shown are from 3 independent experiments.Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Bars shaded in plum indicate moderately pulsing cells (n = 185) and magenta bars indicate highly pulsing cells (n = 57). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 80% and 100% of the generation time.Single-cell size (E) and growth rate (F) averaged over the lifetime of the cell (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; **p = 0.002; ****p < 0.0001. Cells experiencing more DNA damage (VMR>10) are slightly larger and grow at slower rates.


Figure 6Representative time - lapse image series of the dual reporter of RecA and rRNA expression in exponential phase . Pre - CIP growing ( PG - S ) and arrested ( PA - S ) survivors and resistant ( R ) cells are indicated by arrows . CIP exposure ( 1 µg / ml , 4X - MIC ) and SX ( 0 . 5 µM ) death assay ( blue line ) are indicated . Phase - contrast ( gray ) , Prrn - GFPdes ( green ) , and RecA - mCherrywt ( magenta ) fluorescence are merged . Fluorescence images of each channel are scaled to the brightest frame . Cells were imaged at 3 - hour intervals and numbers indicate days . Scale bar , 5 µm . Representative time traces of cell lineages with different fates . Vertical bars represent fresh 7H9 medium ( white ) , CIP ( yellow ) , and SX ( blue ) . Comparison of growth rate ( C ) , cell size averaged over the lifetime of the cell ( D ) , microcolony size ( E ) before CIP exposure Gray and black lines indicate mean ± SD . The data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney test : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA - mCherrywt both before and after drug exposure . fluorescence range over the lifetime of the cell before CIP exposure ( F ) , and mean fluorescence during CIP exposure ( G ) of cells that died ( n = 174 ) and cells that survived ( n = 164 ) . Gray and black lines indicate mean ± SD . The data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney test : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA - mCherrywt both before and after drug exposure . Single - cell fate as a function of VMR of RecA - mCherrywt fluorescence prior to CIP exposure . Significance by Chi - square test of independence : cells that died ( n = 174 ) , cells that survived ( n = 155 ) , and resistant cells ( n = 9 ) . The data shown are from 2 independent experiments . More survivors arise from the subpopulation that does not experience DNA damage before drug exposure .

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Figure 6Representative time-lapse image series of the dual reporter of RecA and rRNA expression in exponential phase. Pre-CIP growing (PG-S) and arrested (PA-S) survivors and resistant (R) cells are indicated by arrows. CIP exposure (1 µg/ml, 4X-MIC) and SX (0.5 µM) death assay (blue line) are indicated. Phase-contrast (gray), Prrn-GFPdes (green), and RecA-mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Cells were imaged at 3-hour intervals and numbers indicate days. Scale bar, 5 µm.Representative time traces of cell lineages with different fates. Vertical bars represent fresh 7H9 medium (white), CIP (yellow), and SX (blue).Comparison of growth rate (C), cell size averaged over the lifetime of the cell (D), microcolony size (E) before CIP exposure Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.fluorescence range over the lifetime of the cell before CIP exposure (F), and mean fluorescence during CIP exposure (G) of cells that died (n = 174) and cells that survived (n = 164). Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.Single-cell fate as a function of VMR of RecA-mCherrywt fluorescence prior to CIP exposure. Significance by Chi-square test of independence: cells that died (n = 174), cells that survived (n = 155), and resistant cells (n = 9). The data shown are from 2 independent experiments. More survivors arise from the subpopulation that does not experience DNA damage before drug exposure.


Figure 1B Measured free PF - 670 exposure in the brain tissue of mice and NHPs . Drug exposure in NHPs after administration of 10 mpk PF - 670 ( AUC = 3 . 6μMh ) is ~ 7 - fold higher than that in mice given 32 mpk PF - 670 ( AUC = 0 . 5 μMh ) ( mean ± SEM ) . C , D Double - plotted actograms of NHPs ' activity for 16 days . NHPs were treated with 10 mpk PF - 670 for 3 consecutive days at the same solar time of day : 4 pm for the DD experiment ( C ) and 4 : 30 pm ( ZT11 ) for the LD experiment ( D ) , respectively , which are highlighted as red lines . White and black rectangles indicate the times of light going on and off ( LD 12 : 12 ) . E , F Phase delay induced by the 3 - day dosing under DD ( E ) or LD ( F ) . The phase of activity onset in NHPs ( 10 mpk ) is more delayed than that in mice ( 32 mpk ) under DD ( E ) , but not under LD ( F ) ( P = 0 . 03 , one - way analysis of variance ( ANOVA ) ; n . s . , no significant difference ) . Veh denotes vehicle . The error bars represent mean ± SEM . The mouse data are adopted from ( Kim et al , 2013 ) . G The quantification of ( E ) and ( F ) indicates that light has a stronger attenuating effect on the PF - 670 - induced phase delay in NHPs than in mice ( P = 0 . 036 , two - way ANOVA ) .

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Figure 1B Measured free PF-670 exposure in the brain tissue of mice and NHPs. Drug exposure in NHPs after administration of 10 mpk PF-670 (AUC=3.6μMh) is ~7-fold higher than that in mice given 32 mpk PF-670 (AUC=0.5 μMh) (mean±SEM).C, D Double-plotted actograms of NHPs' activity for 16 days. NHPs were treated with 10 mpk PF-670 for 3 consecutive days at the same solar time of day: 4 pm for the DD experiment (C) and 4:30 pm (ZT11) for the LD experiment (D), respectively, which are highlighted as red lines. White and black rectangles indicate the times of light going on and off (LD 12:12).E, F Phase delay induced by the 3-day dosing under DD (E) or LD (F). The phase of activity onset in NHPs (10 mpk) is more delayed than that in mice (32 mpk) under DD (E), but not under LD (F) (P=0.03, one-way analysis of variance (ANOVA); n.s., no significant difference). Veh denotes vehicle. The error bars represent mean±SEM. The mouse data are adopted from (Kim et al, 2013). G The quantification of (E) and (F) indicates that light has a stronger attenuating effect on the PF-670-induced phase delay in NHPs than in mice (P=0.036, two-way ANOVA).


Figure 2C Double - plotted actogram of NHPs ' activity for 16 days . The light is on at 6 am and off at 6 pm except for Days 8 to 10 when the light is on at 8 am and off at 8 pm ( i . e . LD is delayed by 2 h ) . D The model successfully predicts the phase shift induced by 2 h jet lag ( Days 8 - 10 ) and its recovery after jet lag ( Days 11 - 13 ) ( n = 7 ; mean ± SEME NHPs were treated with 10 mpk PF - 670 for 3 consecutive days at ZT4 in LD 12 : 12 , which are highlighted as the red line .

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Figure 2C Double-plotted actogram of NHPs' activity for 16 days. The light is on at 6 am and off at 6 pm except for Days 8 to 10 when the light is on at 8 am and off at 8 pm (i.e. LD is delayed by 2 h).D The model successfully predicts the phase shift induced by 2 h jet lag (Days 8-10) and its recovery after jet lag (Days 11-13) (n=7; mean±SEME NHPs were treated with 10 mpk PF-670 for 3 consecutive days at ZT4 in LD 12:12, which are highlighted as the red line.


Figure 3B The simulated PRCs of diurnal NHPs have a higher ratio of maximal magnitudes between advance and delay zones ( A / D = 1 . 25 ) than that of the experimentally measured nocturnal mouse PRC ( A / D = 0 . 41 Here , the red line and red range represent the mean ± SD of the simulated 12 h light PRCs of the NHP model with different gating and adaptation Similarly , the light PRC of diurnal humans has a higher A / D ratio than that of nocturnal

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Figure 3B The simulated PRCs of diurnal NHPs have a higher ratio of maximal magnitudes between advance and delay zones (A/D=1.25) than that of the experimentally measured nocturnal mouse PRC (A/D=0.41 Here, the red line and red range represent the mean±SD of the simulated 12 h light PRCs of the NHP model with different gating and adaptation Similarly, the light PRC of diurnal humans has a higher A/D ratio than that of nocturnal mice


Figure 4E Consistently , higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF - 670 - induced phase delay which was also captured by the NHP model when 20 mpk dosing was used .

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Figure 4E Consistently, higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF-670-induced phase delay which was also captured by the NHP model when 20 mpk dosing was used.


figf1 ( b ) MAPPIT screen identified RAL - interacting proteins . A MAPPIT array covering close to 8 , 500 preys was screened with RALA - G23V as bait . Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1 . ( c ) MAPPIT assay confirmed interaction between the RAL proteins and USP33 . RALA - G23V or RALB - G23V baits were introduced into HEK293T cells together with the indicated preys . The results are expressed as a mean of normalized luciferase activity ± s . d ( leptin - treated cells versus leptin - untreated cells ) for three independent experiments . Statistics source data can be found in Supplementary Table S1 . ( d , e ) USP33 interacts with both RALA and RALB . At 48 h post - transfection with tagged USP33 and RAL expression constructs , the indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) or anti - HA - agarose , followed by immunoblotting using anti - Flag or anti - HA antibodies . WCL , whole - cell lysate ; IP , immunoprecipitates . ( f ) Endogenous USP33 interacts with RALB . Flag - RALB was immunoprecipitated using anti - Flag ( M2 ) agarose and analysed for co - precipitation with endogenous USP33 using anti - USP33 antibody . Cells expressing USP33 shRNA were used as a negative control . ( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The GST - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody . ( h ) USP33 affects ubiquitylation of RALB , but not RALA . 6xHis - tagged ubiquitin and Flag - RAL - G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA . Ubiquitylated RAL proteins were purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALA or RALB . Uncropped images of blots are shown in Supplementary Fig . S7 . ( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The USP33 - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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figf1(b) MAPPIT screen identified RAL-interacting proteins. A MAPPIT array covering close to 8,500 preys was screened with RALA-G23V as bait. Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1.(c) MAPPIT assay confirmed interaction between the RAL proteins and USP33. RALA-G23V or RALB-G23V baits were introduced into HEK293T cells together with the indicated preys. The results are expressed as a mean of normalized luciferase activity ± s.d (leptin-treated cells versus leptin-untreated cells) for three independent experiments. Statistics source data can be found in Supplementary Table S1.(d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA-agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates.(f) Endogenous USP33 interacts with RALB. Flag-RALB was immunoprecipitated using anti-Flag (M2) agarose and analysed for co-precipitation with endogenous USP33 using anti-USP33 antibody. Cells expressing USP33 shRNA were used as a negative control.(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The GST-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.(h) USP33 affects ubiquitylation of RALB, but not RALA. 6xHis-tagged ubiquitin and Flag-RAL-G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALA or RALB. Uncropped images of blots are shown in Supplementary Fig. S7.(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The USP33-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.


figf2 ( a ) Tandem affinity purification of ubiquitylated RALB . 6xHis - ubiquitin and Flag - RALB - G23V were co - transfected into HEK293T cells , and ubiquitylated RALB was purified using anti - Flag resin followed by Co2 + metal affinity chromatography . Isolated RALB was visualized by immunoblotting using anti - RALB antibody . ( b ) RALB undergoes ubiquitylation on multiple sites . Schematic diagram of the RALB domains ( upper panel ) . RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63 % of the RALB amino acid sequence ( Supplementary Fig . S4a ) . The major identified ubiquitylation sites , Lys 47 and Lys 160 , are located in the effector - interacting domain ( green ) and the G5 box ( red ) , respectively . ( c ) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB . ( ( d ) USP33 regulates RALB ubiquitylation at Lys 47 . ( e ) The GTP - GDP status of RALB affects RALB ubiquitylation at Lys 47 . For c - e , 6xHis - tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA . Ubiquitylated RALB was purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALB . WT , wild type . ( f ) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5 . The indicated Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - SEC5 antibody . g ) Ubiquitylation at Lys 47 impairs RALB binding to EXO84 . Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - EXO84 antibody . ( h ) Ubiquitylation at Lys 47 does not affect RALB activity . The activity of RALB mutants was determined by a RALBP1 - RBD binding assay . ( i ) The Rosetta v3 protocol described in ref . was used for ubiquitin - docking simulations . The plot represents the energies of the produced decoys distributed by the root - mean - square deviation ( RMSD ) of ubiquitin position relative to RALA from an arbitrarily chosen decoy . ( j ) Rosetta modelling of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA - EXO84 - RBD complexes . Superposition of the top ten refined solutions of ubiquitylated - RALA - SEC5 - RBD complex , selected by combined scores of two independent docking simulations of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA ( left ) . Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA - EXO84 complex ( right ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf2(a) Tandem affinity purification of ubiquitylated RALB. 6xHis-ubiquitin and Flag-RALB-G23V were co-transfected into HEK293T cells, and ubiquitylated RALB was purified using anti-Flag resin followed by Co2+ metal affinity chromatography. Isolated RALB was visualized by immunoblotting using anti-RALB antibody.(b) RALB undergoes ubiquitylation on multiple sites. Schematic diagram of the RALB domains (upper panel). RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63% of the RALB amino acid sequence (Supplementary Fig. S4a). The major identified ubiquitylation sites, Lys 47 and Lys 160, are located in the effector-interacting domain (green) and the G5 box (red), respectively.(c) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB. ((d) USP33 regulates RALB ubiquitylation at Lys 47. (e) The GTP-GDP status of RALB affects RALB ubiquitylation at Lys 47. For c-e, 6xHis-tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA. Ubiquitylated RALB was purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALB. WT, wild type.(f) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5. The indicated Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-SEC5 antibody.g) Ubiquitylation at Lys 47 impairs RALB binding to EXO84. Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-EXO84 antibody.(h) Ubiquitylation at Lys 47 does not affect RALB activity. The activity of RALB mutants was determined by a RALBP1-RBD binding assay.(i) The Rosetta v3 protocol described in ref. was used for ubiquitin-docking simulations. The plot represents the energies of the produced decoys distributed by the root-mean-square deviation (RMSD) of ubiquitin position relative to RALA from an arbitrarily chosen decoy.(j) Rosetta modelling of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA-EXO84-RBD complexes. Superposition of the top ten refined solutions of ubiquitylated-RALA-SEC5-RBD complex, selected by combined scores of two independent docking simulations of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA (left). Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA-EXO84 complex (right). Uncropped images of blots are shown in Supplementary Fig. S7.


figf3 ( a ) USP33 does not affect RALB activity . The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector ( V ) or HA - tagged USP33 by RALBP1 - RBD binding assay . ( b ) USP33 modulates interaction between RALB and SEC5 . Flag - tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA , USP33 shRNA , empty vector ( V ) or HA - tagged USP33 . The indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - Flag or anti - SEC5 antibodies . ( c ) USP33 regulates RALB binding to EXO84 . Flag - tagged RALB was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - HA or anti - EXO84 antibody . ( d ) Activated RALB co - localizes with both EXO84 and SEC5 . At 24 h after co - transfection with RALB - G23V - EGFP and HA - EXO84 , HeLa cells were immunostained with anti - HA and anti - SEC5 antibodies . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . ( e ) RALB ubiquitylation controls interaction between EXO84 and SEC5 . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing RALB - G23V or RALB - G23V - K47R . The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti - EXO84 antibody . ( f ) USP33 regulates EXO84 - SEC5 complex formation . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti - EXO84 antibody . ( g ) Suppression of SEC5 affects RALB binding to EXO84 . HA - tagged RALB - G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - EXO84 antibody . For a - c , f , g immunoblotting using antibodies specific for USP33 , anti - Flag ( M2 ) or anti - HA determined the levels of USP33 suppression or overexpression . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf3(a) USP33 does not affect RALB activity. The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector (V) or HA-tagged USP33 by RALBP1-RBD binding assay.(b) USP33 modulates interaction between RALB and SEC5. Flag-tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA, USP33 shRNA, empty vector (V) or HA-tagged USP33. The indicated proteins were then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-SEC5 antibodies.(c) USP33 regulates RALB binding to EXO84. Flag-tagged RALB was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-HA or anti-EXO84 antibody.(d) Activated RALB co-localizes with both EXO84 and SEC5. At 24 h after co-transfection with RALB-G23V-EGFP and HA-EXO84, HeLa cells were immunostained with anti-HA and anti-SEC5 antibodies. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels.(e) RALB ubiquitylation controls interaction between EXO84 and SEC5. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing RALB-G23V or RALB-G23V-K47R. The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti-EXO84 antibody.(f) USP33 regulates EXO84-SEC5 complex formation. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti-EXO84 antibody.(g) Suppression of SEC5 affects RALB binding to EXO84. HA-tagged RALB-G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-EXO84 antibody. For a-c,f,g immunoblotting using antibodies specific for USP33, anti-Flag (M2) or anti-HA determined the levels of USP33 suppression or overexpression. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


figf4 ( a ) Suppression of USP33 facilitates TBK1 - SEC5 complex formation . Flag - tagged SEC5 was introduced into HEK293T cells expressing empty vector ( V ) or HA - tagged RALB - G23V together with GFP shRNA or USP33 shRNA as indicated , and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using antibody specific for TBK1 . ( c ) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB - SEC5 complex in response to poly ( I : C ) treatment . Flag - tagged SEC5 and the indicated HA - tagged RALB mutants were introduced into 293 - hTLR3 cells . At 48 h after transfection cells were treated 100 μg ml − 1 of poly ( I : C ) for 6 h . Flag - tagged SEC5 was immunoprecipitated with anti - Flag ( M2 ) , and analysed for co - precipitation by immunoblotting using anti - HA antibody . ( c ) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB - SEC5 complex in response to poly ( I : C ) treatment . Flag - tagged SEC5 and the indicated HA - tagged RALB mutants were introduced into 293 - hTLR3 cells . At 48 h after transfection cells were treated 100 μg ml − 1 of poly ( I : C ) for 6 h . Flag - tagged SEC5 was immunoprecipitated with anti - Flag ( M2 ) , and analysed for co - precipitation by immunoblotting using anti - HA antibody . ( d ) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3 . At 48 h after transfection with empty vector ( V ) , Flag - RALB - G23V and Flag - RALB - G23V - K47R , 293 - hTLR3 cells were treated with 10 μg ml − 1 of poly ( I : C ) for 6 h . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf4(a) Suppression of USP33 facilitates TBK1-SEC5 complex formation. Flag-tagged SEC5 was introduced into HEK293T cells expressing empty vector (V) or HA-tagged RALB-G23V together with GFP shRNA or USP33 shRNA as indicated, and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using antibody specific for TBK1.(c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.(c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.(d) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3. At 48 h after transfection with empty vector (V), Flag-RALB-G23V and Flag-RALB-G23V-K47R, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


figf5 ( a - d ) USP33 inhibits assembly of SEC5 - beclin - 1 complexes . The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti - Flag ( M2 ) , anti - HA or anti - SEC5 antibody and analysed for co - precipitation by immunoblotting using specific antibodies as indicated . ( e ) USP33 potentiates the interaction between EXO84 and beclin 1 . Endogenous beclin 1 was immunoprecipitated from HEK293T cells , expressing either empty vector ( V ) , HA - USP33 wild type ( WT ) or the catalytically inactive mutant C194S - H683Q , using anti - beclin 1 antibody . Immunoprecipitates were analysed for co - precipitation by immunoblotting using anti - EXO84 or anti - beclin 1 antibodies . ( f , g ) RALB but not RALA undergoes deubiquitylation under nutrient deprivation . 6xHis - tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells . At 48 h after transfection cells were deprived of nutrients and incubated in Hank ' s buffered salt solution ( HBSS ) medium for 90 min . Ubiquitylated RALproteins were purified by Co2 + metal affinity chromatography and analysed by immunoblotting using anti - RALA or anti - RALB antibodies . ( h ) Nutrient starvation results in the accumulation of USP33 . HEK TE cells were incubated in HBSS medium for the indicated periods of time . Immunoblotting was performed using anti - USP33 antibody . ( i ) USP33 co - localizes with RALB - positive vesicles in the absence of nutrients . At 48 h after co - transfection with RALB - G23V - EGFP and USP33 - mCherry , HeLa cells were incubated in HBSS medium for 90 min . Cells were imaged for EGFP and mCherry fluorescence . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . Arrows indicate USP33 co - localization to RALB - positive vesicles . ( j ) Beclin 1 co - localizes with USP33 . HeLa cells were transfected with beclin - 1 - HA and USP33 - mCherry . At 48 h after transfection , cells were incubated in HBSS medium for 90 minutes and treated as in i . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf5(a-d) USP33 inhibits assembly of SEC5-beclin-1 complexes. The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti-Flag (M2), anti-HA or anti-SEC5 antibody and analysed for co-precipitation by immunoblotting using specific antibodies as indicated.(e) USP33 potentiates the interaction between EXO84 and beclin 1. Endogenous beclin 1 was immunoprecipitated from HEK293T cells, expressing either empty vector (V), HA-USP33 wild type (WT) or the catalytically inactive mutant C194S-H683Q, using anti-beclin 1 antibody. Immunoprecipitates were analysed for co-precipitation by immunoblotting using anti-EXO84 or anti-beclin 1 antibodies.(f,g) RALB but not RALA undergoes deubiquitylation under nutrient deprivation. 6xHis-tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells. At 48 h after transfection cells were deprived of nutrients and incubated in Hank's buffered salt solution (HBSS) medium for 90 min. Ubiquitylated RALproteins were purified by Co2+ metal affinity chromatography and analysed by immunoblotting using anti-RALA or anti-RALB antibodies.(h) Nutrient starvation results in the accumulation of USP33. HEK TE cells were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using anti-USP33 antibody.(i) USP33 co-localizes with RALB-positive vesicles in the absence of nutrients. At 48 h after co-transfection with RALB-G23V-EGFP and USP33-mCherry, HeLa cells were incubated in HBSS medium for 90 min. Cells were imaged for EGFP and mCherry fluorescence. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Arrows indicate USP33 co-localization to RALB-positive vesicles.(j) Beclin 1 co-localizes with USP33. HeLa cells were transfected with beclin-1-HA and USP33-mCherry. At 48 h after transfection, cells were incubated in HBSS medium for 90 minutes and treated as in i. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S7.


figf6 ( a ) USP33 depletion inhibits accumulation of LC3 - lipid conjugates . HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h , in the presence or absence of 50 μM of chloroquine ( CQ ) . Cells were then assayed for the relative accumulation of LC3 - I and LC3 - II by immunoblotting analysis using anti - LC3 antibody . LC3 - II / GAPDH ratios have been calculated using ImageJ densitometric analysis . ( b - d ) USP33 expression levels affect the accumulation of LC3punctae . Wild - type USP33 or a USP33 - shRNA - resistant USP33 mutant ( USP33R ) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA . The generated cells were incubated in HBSS medium for 4 h . USP33 was analysed by immunoblotting using anti - USP33 antibody ( b ) . Representative images of LC3immunostaining are shown ( c ) . Endogenous LC3punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software . The mean distribution of LC3punctae per cell is represented as the mean of two independent experiments . Statistics source data for d can be found in Supplementary Table S1 ( d ) . ( e ) USP33 depletion leads to increased levels of p62 . HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time . Immunoblotting was performed using the indicated antibodies . ( f ) Overexpression of the RALB - G23V - K47R mutant leads to accumulation of LC3 - lipid conjugates . HEK293T cells overexpressing the RALB - G23V or RALB - G23V - K47R mutants were assayed for the relative accumulation of LC3 - I and LC3 - II by immunoblotting using anti - LC3 antibody .

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figf6(a) USP33 depletion inhibits accumulation of LC3-lipid conjugates. HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h, in the presence or absence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting analysis using anti-LC3 antibody. LC3-II/GAPDH ratios have been calculated using ImageJ densitometric analysis.(b-d) USP33 expression levels affect the accumulation of LC3punctae. Wild-type USP33 or a USP33-shRNA-resistant USP33 mutant (USP33R) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA. The generated cells were incubated in HBSS medium for 4 h. USP33 was analysed by immunoblotting using anti-USP33 antibody (b). Representative images of LC3immunostaining are shown (c). Endogenous LC3punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software. The mean distribution of LC3punctae per cell is represented as the mean of two independent experiments. Statistics source data for d can be found in Supplementary Table S1 (d).(e) USP33 depletion leads to increased levels of p62. HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using the indicated antibodies.(f) Overexpression of the RALB-G23V-K47R mutant leads to accumulation of LC3-lipid conjugates. HEK293T cells overexpressing the RALB-G23V or RALB-G23V-K47R mutants were assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting using anti-LC3 antibody.


Figure 1 ( A ) Western Blot detection of ATM pS1981 , SMCHD1 , TRF2 and hnRNPA1 loading control in wild - type and two SMCHD1 knockout ( KO1 and KO2 ) HeLa cells transfected with shTRF2 plasmid or EV control . ( B ) Representative metaphase spreads from wild - type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control . Telomeric signals were detected with Cy3 - OO - ( CCCTAA ) 3 and are false colored in red , DNA is stained with DAPI and is false colored in cyan . Scale bar : 5µm . ( C ) Quantification of telomere fusions from experiment Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 3 , 000 telomeres counted / condition / experiment ) . ( * ) P < 0 . 05 ; unpaired two - tailed Student ' s t - test . ( D ) Representative cell cycle profiles from two biological replicates of WT and SMCHD1 KO HeLa cells transfected with shTRF2 plasmid or EV control . Cells were stained with propidium iodide and analyzed by Flow cytometry .

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Figure 1(A) Western Blot detection of ATM pS1981, SMCHD1, TRF2 and hnRNPA1 loading control in wild-type and two SMCHD1 knockout (KO1 and KO2) HeLa cells transfected with shTRF2 plasmid or EV control.(B) Representative metaphase spreads from wild-type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control. Telomeric signals were detected with Cy3-OO-(CCCTAA)3 and are false colored in red, DNA is stained with DAPI and is false colored in cyan. Scale bar: 5µm.(C) Quantification of telomere fusions from experiment Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>3,000 telomeres counted/condition/experiment). (*) P < 0.05; unpaired two-tailed Student's t-test.(D) Representative cell cycle profiles from two biological replicates of WT and SMCHD1 KO HeLa cells transfected with shTRF2 plasmid or EV control. Cells were stained with propidium iodide and analyzed by Flow cytometry.


Figure 2 ( A ) Western Blot detection of SMCHD1 , TRF2 and hnRNPA1 in SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days ( d7 , d8 , d11 ) . ( B ) Quantification of telomere fusions in SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days ( d7 , d8 , d11 ) . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 1 , 900 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 , ( * * ) P < 0 . 01 ; ( * ) P < 0 . 05 ; unpaired two - tailed Student ' s t - test . ( C ) Terminal Restriction Fragment ( TRF ) analysis of telomeric DNA to detect 3 ' overhang processing of genomic DNA isolated from SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 cells treated as in the experiment in ( B ) . ( Left ) Radiolabeled ( CCCTAA ) n probe was hybridized with a short run ( upper panel ) and long run ( lower panel ) native DNA gel to detect the signal of the telomeric 3 ' overhang . Samples used for the short and the long run were from the same digestion split into two . Exo I treatment ( + Exo ) was used as a control that single stranded telomeric signal was terminal . ( Right ) The total TTAGGG signal in the same lane was deteced upon denaturation and hybridization with the same probe . ( D ) Quantification of the telomeric overhang signal at d11 after doxycyclin addition to SMCHD1 wild - type and SMCHD1 knockout HeLa shTRF2 inducible cells . The bar graph represents the average overhang signal intensity from two biological replicates as percentage of the signal in the cells untreated with doxycycline with error bars representing the standard deviation ( SD ) of the sample .

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Figure 2(A) Western Blot detection of SMCHD1, TRF2 and hnRNPA1 in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11).(B) Quantification of telomere fusions in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11). Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>1,900 telomeres counted/condition/experiment). (***) P < 0.001, (**) P< 0.01; (*) P< 0.05; unpaired two-tailed Student's t-test.(C) Terminal Restriction Fragment (TRF) analysis of telomeric DNA to detect 3'overhang processing of genomic DNA isolated from SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated as in the experiment in (B). (Left) Radiolabeled (CCCTAA)n probe was hybridized with a short run (upper panel) and long run (lower panel) native DNA gel to detect the signal of the telomeric 3' overhang. Samples used for the short and the long run were from the same digestion split into two. Exo I treatment (+ Exo) was used as a control that single stranded telomeric signal was terminal. (Right) The total TTAGGG signal in the same lane was deteced upon denaturation and hybridization with the same probe.(D) Quantification of the telomeric overhang signal at d11 after doxycyclin addition to SMCHD1 wild-type and SMCHD1 knockout HeLa shTRF2 inducible cells. The bar graph represents the average overhang signal intensity from two biological replicates as percentage of the signal in the cells untreated with doxycycline with error bars representing the standard deviation (SD) of the sample.


Figure 3A Western blot detection of ATM pS1981 , SMCHD , TRF2 , γH2AX , and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and EV control . B Representative images for CO - FISH on metaphase spreads in wild type ( WT ) Hela and U2OS cells . The leading strand telomeres were labeled by PNA - FISH with a ( TTAGGG ) 3 - Alexa488 probe ( green ) , the lagging strand with a ( CCCTAA ) 3 - Cy5 probe ( pink ) , and the DNA was stained with DAPI . Examples of telomere sister chromatid exchange ( t - SCEs ) events are marked with white arrows . Scale bar : 5µmC Quantification of chromosome ends with t - SCEs detected by CO - FISH . Data represent means of 2 independent experiments ± SD ( > 700 telomeres / condition / experiment ) with individual average values indicated .

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Figure 3A Western blot detection of ATM pS1981, SMCHD, TRF2, γH2AX, and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and EV control.B Representative images for CO-FISH on metaphase spreads in wild type (WT) Hela and U2OS cells. The leading strand telomeres were labeled by PNA-FISH with a (TTAGGG)3-Alexa488 probe (green), the lagging strand with a (CCCTAA)3-Cy5 probe (pink), and the DNA was stained with DAPI. Examples of telomere sister chromatid exchange (t-SCEs) events are marked with white arrows. Scale bar: 5µmC Quantification of chromosome ends with t-SCEs detected by CO-FISH. Data represent means of 2 independent experiments± SD (>700 telomeres/condition/experiment) with individual average values indicated.


Figure 4 ( A - C ) Representative images for detection of ATM pS9181 , γH2AX , and 53BP1 at telomeres in wild - type ( WT ) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector ( EV ) control . Immunofluoresence ( IF ) for ATM pS1981 ( gray ) , γH2AX ( green ) and 53BP1 ( yellow ) was combined with telomeric ( CCCTAA ) 3 - FISH ( red ) and DAPI staining total DNA . Scale bars : 5µm ( D ) Quantification of the number of cells containing > 5 Telomere dysfunction Induced Foci ( TIFs ) detected Data represent the mean of 4 independent experiments ± SD ( > 200 cells / condition / experiment ) for ATM pS1981 and 3 independent experiments ± SD ( > 200 cells / condition / experiment ) for γH2AX and 53BP1 . ( * * * ) P < 0 . 001 ; unpaired two - tailed Student ' s t - test . ( E ) Quantification of the number of 53BP1 TIFs per cell detected from three independent experiments . Data represent individual values per cell with the bar representing mean ± SD . ( * * * ) P < 0 . 001 ; unpaired two - tailed Student ' s t - test .

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Figure 4(A-C) Representative images for detection of ATM pS9181, γH2AX, and 53BP1 at telomeres in wild-type (WT) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector (EV) control. Immunofluoresence (IF) for ATM pS1981 (gray), γH2AX (green) and 53BP1 (yellow) was combined with telomeric (CCCTAA)3-FISH (red) and DAPI staining total DNA. Scale bars: 5µm(D) Quantification of the number of cells containing >5 Telomere dysfunction Induced Foci (TIFs) detected Data represent the mean of 4 independent experiments ± SD (>200 cells/condition/experiment) for ATM pS1981 and 3 independent experiments ± SD (>200 cells/condition/experiment) for γH2AX and 53BP1. (***) P < 0.001; unpaired two-tailed Student's t-test.(E) Quantification of the number of 53BP1 TIFs per cell detected from three independent experiments. Data represent individual values per cell with the bar representing mean ± SD. (***) P < 0.001; unpaired two-tailed Student's t-test.


Figure 5 ( A ) Western blot detection of SMCHD1 , ATMpS1981 , CHK2 pT68 , CHK1 pS345 , RPA pS33 , RPA pS4 / 8 , γH2AX and Vinculin in wild - type or SMCHD1 KO HeLa cells γ - irradiated with 3Gy dose and harvested at the indicated times ( 1h , 12h , 24h ) after the treatment . ( B ) Representative images for Immunofluoresence ( IF ) detection of ATMpS1981 in wild - type HeLa cells γ - irradiated with a dose of 3Gy and harvested after the indicated times . Scale bar : 5µm ( C ) Quantification of the average number of ATMpS1981 foci per cell detected as in ( B ) in wild - type and SMCHD1 KO γ - irradiated HeLa cells . Data represent mean of 3 independent experiments ± SD ( > 200 cells / condition / experiment ) with individual average values indicated .

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Figure 5(A) Western blot detection of SMCHD1, ATMpS1981, CHK2 pT68, CHK1 pS345, RPA pS33, RPA pS4/8, γH2AX and Vinculin in wild-type or SMCHD1 KO HeLa cells γ-irradiated with 3Gy dose and harvested at the indicated times (1h, 12h, 24h) after the treatment.(B) Representative images for Immunofluoresence (IF) detection of ATMpS1981 in wild-type HeLa cells γ-irradiated with a dose of 3Gy and harvested after the indicated times. Scale bar: 5µm(C) Quantification of the average number of ATMpS1981 foci per cell detected as in (B) in wild-type and SMCHD1 KO γ-irradiated HeLa cells. Data represent mean of 3 independent experiments ± SD (>200 cells/condition/experiment) with individual average values indicated.


Figure 6 ( A ) Western Blot detection of SMCHD1 , TRF2 , TPP1 and hnRNPA1 in wild - type or SMCHD1 knockout HeLa cells transfected with the indicated shRNAs ( shTRF2 , shTPP1 , shTRF2 / shTPP1 ) or EV control . ( B ) Representative metaphase spreads ( Scale bar : 5µm ) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 2 , 500 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 ; ( * * ) P < 0 . 01 unpaired two - tailed Student ' s t - test . ( C ) Representative metaphase spreads ( Scale bar : 5µm ) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi ( VE - 821 ) and quantification of telomere fusions . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 2 , 000 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 ; ( * * ) P < 0 . 01 ; ( * ) P < 0 . 05 unpaired two - tailed Student ' s t - test .

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Figure 6(A) Western Blot detection of SMCHD1, TRF2, TPP1 and hnRNPA1 in wild-type or SMCHD1 knockout HeLa cells transfected with the indicated shRNAs (shTRF2, shTPP1, shTRF2/shTPP1) or EV control.(B) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,500 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01 unpaired two-tailed Student's t-test.(C) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi (VE-821) and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,000 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01; (*) P < 0.05 unpaired two-tailed Student's t-test.


Figure 3The number of neoblasts did not change significantly following inhibition of the MTC - gene kiaa1429 . Shown is FISH of smedwi - 1 ( A ) in control and kiaa1429 ( RNAi ) animals ( left ) . smedwi - 1 + cells were counted following FISH and showed no significant difference in expression ( right ; bar represents average normalized cell count ; Materials and Methods ) . Scale = 100 µm . The number of neoblasts did not change significantly following inhibition of the MTC - gene kiaa1429 . Shown is FISH of h2b ( B ) in control and kiaa1429 ( RNAi ) animals ( left ) . h2b + cells were counted following FISH and showed no significant difference in expression ( right ; bar represents average normalized cell count ; Materials and Methods ) . Scale = 100 µm . ( E ) Representative FACS plots of cells isolated from control ( top ) and kiaa1429 ( RNAi ) animals ( bottom ) show an increase in cell abundance in the X2 ( Fincher et al , 2018 ) gate ( green ; Materials and Methods ) . ( F ) Quantification of the cells in each FACS gate in control and kiaa1429 ( RNAi ) animals showed a six - fold increase in the abundance of cells in the X2 gate ( Student ' s t - test * * * - p < 0 . 005 . FACS experiments were performed in biological triplicates . Boxes represent the IQR , whiskers represent the 1 . 5 x IQR , and central band represents the

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Figure 3The number of neoblasts did not change significantly following inhibition of the MTC-gene kiaa1429. Shown is FISH of smedwi-1 (A) in control and kiaa1429 (RNAi) animals (left). smedwi-1+ cells were counted following FISH and showed no significant difference in expression (right; bar represents average normalized cell count; Materials and Methods). Scale = 100 µm.The number of neoblasts did not change significantly following inhibition of the MTC-gene kiaa1429. Shown is FISH of h2b (B) in control and kiaa1429 (RNAi) animals (left). h2b+ cells were counted following FISH and showed no significant difference in expression (right; bar represents average normalized cell count; Materials and Methods). Scale = 100 µm.(E) Representative FACS plots of cells isolated from control (top) and kiaa1429 (RNAi) animals (bottom) show an increase in cell abundance in the X2 (Fincher et al, 2018) gate (green; Materials and Methods). (F) Quantification of the cells in each FACS gate in control and kiaa1429 (RNAi) animals showed a six-fold increase in the abundance of cells in the X2 gate (Student's t-test *** - p < 0.005. FACS experiments were performed in biological triplicates. Boxes represent the IQR, whiskers represent the 1.5 x IQR, and central band represents the med


figf1 ( b ) Coimmuno - precipitation ( IP ) of Fbxo7 - HA and Flag - Parkin in whole - cell lysates from U2OS cells over - expressing both proteins . IB , immunoblot . Molecular mass markers are in kilodaltons . ( c ) Coimmunoprecipitation of endogenous Fbxo7 with Flag - Parkin in HEK293T cells transfected with Flag - Parkin or a control protein ( EGFP ) . ( d ) Flag immunoprecipitation was repeated in U2OS cells transfected with Flag - Parkin and either full - length ( 1 - 522 ) T7 - Fbxo7 or an N - terminal truncation lacking the Ubl domain ( 89 - 522 ) . ( e ) As in d , using lysates from U2OS cells expressing Flag - Parkin and T7 - Fbxo7 , either full length ( 1 - 522 ) or a C - terminal deletion of the proline - rich region ( 1 - 398 ) . * indicates IgG heavy chain . ( f ) In vitro - translated Flag - Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain ( 1 - 88 ) immobilized on glutathione beads . Bead - bound proteins and inputs were analyzed by immunoblotting with anti - Parkin antibodies . ( g ) The disease - causing mutation T22M interferes with Fbxo7 ' s interaction with Parkin . Coimmunoprecipitation was performed as in b using lysates from U2OS cells expressing Flag - Parkin and WT or T22M Fbxo7 - HA . ( h ) Coimmunoprecipitation of Flag - Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins . CxVα and GAPDH are markers for mitochondria and cytoplasm , respectively . All western blots are representative of experiments performed at least three times . Full - length blots are presented in Supplementary Figure 9 .

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figf1(b) Coimmuno-precipitation (IP) of Fbxo7-HA and Flag-Parkin in whole-cell lysates from U2OS cells over-expressing both proteins. IB, immunoblot. Molecular mass markers are in kilodaltons.(c) Coimmunoprecipitation of endogenous Fbxo7 with Flag-Parkin in HEK293T cells transfected with Flag-Parkin or a control protein (EGFP).(d) Flag immunoprecipitation was repeated in U2OS cells transfected with Flag-Parkin and either full-length (1-522) T7-Fbxo7 or an N-terminal truncation lacking the Ubl domain (89-522).(e) As in d, using lysates from U2OS cells expressing Flag-Parkin and T7-Fbxo7, either full length (1-522) or a C-terminal deletion of the proline-rich region (1-398). * indicates IgG heavy chain.(f) In vitro-translated Flag-Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain (1-88) immobilized on glutathione beads. Bead-bound proteins and inputs were analyzed by immunoblotting with anti-Parkin antibodies.(g) The disease-causing mutation T22M interferes with Fbxo7's interaction with Parkin. Coimmunoprecipitation was performed as in b using lysates from U2OS cells expressing Flag-Parkin and WT or T22M Fbxo7-HA.(h) Coimmunoprecipitation of Flag-Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins. CxVα and GAPDH are markers for mitochondria and cytoplasm, respectively. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.