F . Immunoblot of ASC oligomerization in cross - linked pellets ( insoluble ) whole lysate ( soluble ) in HEK293T transfected with His - NLRP3 TRIM50 or TRIM50 coiled - coil domain - deleted mutants . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . Data are representative of three biological replicates with similar results .

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

F. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate(soluble) in HEK293T cells transfected with His-NLRP3 and TRIM50 or TRIM50 coiled-coil domain-deleted mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


A . Survival status of WT 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 ±

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

A. 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 ± SD


B . ELISA of IL - 1β , TNF - α 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 - Student ' s t - Data are representative of three biological replicates with similar results . ( mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ± SD


C . Histopathological of lung tissues by hematoxylin eosin ( H & E ) . 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 - Student ' s t - Data are representative of three biological replicates with similar results . ( mean ±

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

C. 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 ± SD


D . Flow cytometry of neutrophils ( left ) 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 - Student ' s t - Data are representative of three biological replicates with similar results . ( mean ±

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

D. 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 ± SD


E . WT or Trim50 - / - mice were i . p . injected with FA for 36h , followed with immunoblot of NLRP3 , pro - IL - 1β , Caspase1 p20 , IL - 1β in kidney tissue samples . Data information : P - values are shown , two - way ANOVA ( A ) or two - Student ' s t - Data are representative of three biological replicates with similar results . ( mean ±

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

E. 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 ± SD


F . H & E of kidney tissue sections . Scale bars : 20μm .

F. H&E staining of kidney tissue sections. Scale bars: 20μm.


G . Periodic - acid - Schiff ( PAS ) of kidney tissue sections . Scale bars : 20μm .

G. Periodic-acid-Schiff (PAS) staining of kidney tissue sections. Scale bars: 20μm.


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

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

Representative 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.


Color - coded single - time traces of four reporters expressed as of generation time ( B ) . Population coefficients of variation of ( % ) are in color - coded boxes . Single - VMR of ( C ) averaged over lifetime of ( D ) in exponential phase . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis Dunn ' s multiple comparison : ns , not significant ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 . data shown are from 2 independent experiments ( n = 105 per ) . Single - decreases but variation does not change in strains devoid of positive regulation . Single - increases variation decreases in devoid of negative regulation .

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

Color-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 ( left panel ) averaged for 50 per ( mean ± SD ) treated with MMC ( 0 . 5 μg / ml ) for 2 hours ( gray shading ) . induction during 6 hours from MMC exposure ( right panel ) . Asterisks denote significance by Kruskal - Wallis Dunn ' s multiple comparison : ns , not significant ; * * * * p < 0 . 0001 . data shown are from 2 independent experiments .

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

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 - of independent lineages separated by black vertical lines . Color - coded traces represent single - of random descendants monitored in fresh 7H9 medium for 24 hours ( n = 20 ) . gray vertical bands represents divisions . data shown are from 2 independent experiments .

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

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 profiles averaged for each reporter ( color - coded as in A ) . Error bars are SD ( 4 < n < 37 ) . data shown are from 2 independent experiments . Pulses are abrogated in devoid of negative regulation .

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

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.


of characteristics . Black lines indicate means SD ( 52 < n < 88 ) . Significance by Kruskal - Wallis nd Dunn ' s multiple comparison : ns , not significant ; * * * * p < 0 . 0001 . data shown are from 2 independent experiments .

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

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 distribution of peak time of expressed as a of generation time ( 52 < n < 88 ) . Different strains are color - coded . Statistical by one - way ANOVA Holm - Sidak ' s multiple comparisons . Pulses are weaker fewer in devoid of positive regulation but are not displaced relative to generation time .

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

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.


Spearman ' s correlation between green ( A - D ) or yellow ( E ) range red range over lifetime of ( 105 < n < 153 ) . data shown are from 2 independent experiments . On x - axes P2P1recA - GFPdes ( A - D ) or a C - term translational reporter of SSBa fused to mCitrinewt ( E ) . On y - axes , a C - term translational reporter of RecA fused to mCherrywt ( A E ) , transcriptional reporters of ruvC ( B ) radA ( C ) , mCherrydes expressed from a constitutive control promoter ( D ) . Colors ( A - D ) indicate subpopulations segregated by VMR of P2P1recA - GFPdes : gray ( 0 < VMR ≤ 1 ) , green ( 1 < VMR ≤ 10 ) , green ( VMR > 10 ) . Colors ( E ) indicate subpopulations segregated by VMR of RecA - mCherrywt : gray ( 0 < VMR ≤ 1 ) , plum ( 1 < VMR ≤ 10 ) , magenta ( VMR > 10 ) .

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

Spearman'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 ) red , measured 3 hours after MMC exposure ( 203 < n < 314 ) . data shown are from 2 independent experiments . On x - axes P2P1recA - GFPdes ( F - I ) or a C - term translational reporter of SSBa fused to mCitrinewt ( J ) . On y - axes , a C - term translational reporter of RecA fused to mCherrywt ( F J ) , transcriptional reporters of ruvC ( G ) radA ( H ) , mCherrydes expressed from a constitutive control promoter ( I ) . Positive correlations between recA other - damage markers both under normal growth conditions , where spontaneous - damage events occur at subpopulation level , in presence of an alkylating agent that induces double - strand breaks in entire population , validate recA as a reliable proxy for damage in single .

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

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.


Representative time - lapse image series of merodiploid recA reporter ( P2P1recA - GFPdes _ RecA - mCherrywt ) cultured in fresh ( A ) or spent ( B ) flow medium ( see Materials Methods ) . Fresh 7H9 medium is unlabeled ; spent medium ( S ) , ciprofloxacin 1 µg / ml in fresh ( CIP ) or spent ( S - CIP ) medium , SYTOX 0 . 5 µM ( SX ) are labeled . Phase - contrast ( gray ) , GFPdes ( green ) , mCherrywt ( magenta ) are merged . of each channel are scaled to brightest frame . - green background in first 4 frames ( B ) is due to autofluorescence of spent medium . were imaged at 30 - min intervals numbers represent hours . Scale bar , 10 µm .

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

Representative 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 lineages of fed with fresh medium ( C ) or spent medium ( D ) . Different styles are used for lineages with different fates : lysis ( L ) ; SYTOX - positive ( SX + ) ; survival ( S ) . breaks represent division events in same lineage , where a single random daughter is tracked for clarity . Vertical bars represent fresh ( ) or spent ( shading ) 7H9 medium , CIP ( yellow ) , SX ( blue ) .

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

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 - growth rates prior to CIP exposure for " D " that die " S " that survive regrow after CIP washout ( E ) , lag time of regrowth ( F ) . were cultured in fresh ( EXP ) or spent ( STAT ) flow medium . Gray black lines indicate mean ± SD . Significance by Kruskal - Wallis Dunn ' s multiple comparison ( E ) unpaired Mann - Whitney ( F ) : ns , not significant ; * * * * p < 0 . 0001 ; 6 < n < 84 . data shown are from 2 independent experiments . Single - fate is independent of growth rate before exposure to drug . However , cultured in spent medium ( STAT ) grow at significantly slower rates , survive at higher frequencies , recover faster after CIP washout compared to cultured in fresh medium ( EXP ) .

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

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 ) mCherrywt ( H ) time - traces for single - lineages of cultured in fresh medium ( EXP ) cultured in spent medium ( STAT ) , having different fates . Vertical bars are color - coded as in C D .

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

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 - fate as a function of VMR of P2P1recA - GFPdes ( I ) RecA - mCherrywt ( J ) prior to CIP exposure . Significance by Chi - square of independence for that die ( n = 84 ) that survive ( n = 54 ) . data shown are from 2 independent experiments . that do not experience damage prior to drug exposure ( RecA - non - pulsing ) survive more than that do experience damage ( RecA - pulsing ) .

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

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).


Spearman ' s correlation between single - RecA - mCherrywt range VMR over lifetime of ( n = 360 ) . data shown are from 3 independent experiments . Dashed lines separate different of dispersion , colors denote subpopulations that are underdispersed ( gray ) , dispersed ( plum ) , or highly dispersed ( magenta ) . RecA - mCherrywt of subpopulations segregated by VMR ( 58 < n < 159 ) . data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significant difference by Kruskal - Wallis Dunn ' s multiple comparison : * * * * p < 0 . 0001 .

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

Spearman'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 - time traces of RecA - mCherrywt expressed as of generation time of subpopulations segregated by VMR . average ( solid black lines ) half of generation time ( dashed lines ) are indicated . Population statistics ( n = 360 ) : 40 % ( 0 < VMR ≤ 1 ) ; 44 % ( 1 < VMR ≤ 10 ) ; 16 % ( VMR > 10 ) . data shown are from 3 independent experiments .

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

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 distribution of peak time expressed as a of generation time . Bars shaded in plum indicate moderately pulsing ( n = 185 ) magenta bars indicate highly pulsing ( n = 57 ) . Significance by Mann - Whitney . data shown are from 3 independent experiments . Peaks mainly occur between 80 % 100 % of generation time .

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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 - ( E ) growth rate ( F ) averaged over lifetime of ( 58 < n < 159 ) . data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis Dunn ' s multiple comparison : ns , not significant ; * * p = 0 . 002 ; * * * * p < 0 . 0001 . experiencing more damage ( VMR > 10 ) are slightly larger grow at slower rates .

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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.


Representative time - lapse image series of dual reporter of RecA rRNA expression in exponential phase . Pre - CIP growing ( PG - S ) arrested ( PA - S ) survivors resistant ( R ) are indicated by arrows . CIP exposure ( 1 µg / ml , 4X - MIC ) SX ( 0 . 5 µM ) ( blue ) are indicated . Phase - contrast ( gray ) , Prrn - GFPdes ( green ) , RecA - mCherrywt ( magenta ) are merged . of each channel are scaled to brightest frame . were imaged at 3 - hour intervals numbers indicate days . Scale bar , 5 µm .

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Representative 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 lineages with different fates . Vertical bars represent fresh 7H9 medium ( ) , CIP ( yellow ) , SX ( blue ) .

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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 ) , averaged over lifetime of ( D ) , microcolony ( E ) before CIP exposure Gray black lines indicate mean ± SD . data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin lower expression of RecA - mCherrywt both before after drug exposure .

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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.


range over lifetime of before CIP exposure ( F ) , mean during CIP exposure ( G ) of that died ( n = 174 ) that survived ( n = 164 ) . Gray black lines indicate mean ± SD . data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin lower expression of RecA - mCherrywt both before after drug exposure .

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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 - fate as a function of VMR of RecA - mCherrywt prior to CIP exposure . Significance by Chi - square of independence : that died ( n = 174 ) , that survived ( n = 155 ) , resistant ( n = 9 ) . data shown are from 2 independent experiments . More survivors arise from subpopulation that does not experience damage before drug exposure .

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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.


B Measured free PF - 670 exposure in brain tissue of mice 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 ± ) .

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B 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 same solar time of day : 4 pm for DD experiment ( C ) 4 : 30 pm ( ZT11 ) for LD experiment ( D ) , respectively , which are highlighted as red lines . black rectangles indicate times of going on off ( LD 12 : 12 ) .

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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 3 - day dosing under DD ( E ) or LD ( F ) . 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 of variance ( ANOVA ) ; n . s . , no significant difference ) . Veh denotes vehicle . error bars represent mean ± . mouse data are adopted from ( Kim et al , 2013 ) . G of ( E ) ( F ) indicates that has a stronger attenuating effect on PF - 670 - induced phase delay in NHPs than in mice ( P = 0 . 036 , two - way ANOVA ) .

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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).


C Double - plotted actogram of NHPs ' activity for 16 days . is on at 6 am off at 6 pm except for Days 8 to 10 when is on at 8 am off at 8 pm ( i . e . LD is delayed by 2 h ) .

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C 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 model successfully predicts phase shift induced by 2 h jet lag ( Days 8 - 10 ) its recovery after jet lag ( Days 11 - 13 ) ( n = 7 ; mean ±

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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±SEM


E NHPs were treated with 10 mpk PF - 670 for 3 consecutive days at ZT4 in LD 12 : 12 , which are highlighted as red .

E 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.


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

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B 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


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

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E 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.


( b ) MAPPIT screen identified RAL - interacting . A MAPPIT array covering close to 8 , 500 preys was screened with RALA - G23V as bait . Data are shown in a volcano plot of log of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1 .

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(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 confirmed interaction between RAL USP33 . RALA - G23V or RALB - G23V baits were introduced into HEK293T together with indicated preys . results are expressed as a mean of normalized luciferase activity ± s . d ( leptin - treated versus leptin - untreated ) for three independent experiments . Statistics source data can be found in Supplementary Table S1 .

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(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 RALB . At 48 h post - transfection with tagged USP33 RAL expression constructs , indicated were then immunoprecipitated with anti - Flag ( M2 ) or anti - HA - agarose , followed by immunoblotting using anti - Flag or anti - HA antibodies . WCL , whole - lysate ; IP , immunoprecipitates .

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(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 analysed for co - precipitation with endogenous USP33 using anti - USP33 antibody . expressing USP33 shRNA were used as a negative control .

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(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 RAL . GST - tagged RALA RALB were isolated with glutathione - agarose mixed with lysates derived from HEK293T expressing Flag - USP33 in presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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(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 Flag - RAL - G23V mutants were introduced into HEK293T stably expressing GFP shRNA or USP33 shRNA . Ubiquitylated RAL were purified by Co2 + metal affinity chromatography detected by antibodies specific to RALA or RALB . Uncropped of blots are shown in Supplementary Fig . S7 .

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(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 RAL . USP33 - tagged RALA RALB were isolated with glutathione - agarose mixed with lysates derived from HEK293T expressing Flag - USP33 in presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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(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.


( a ) Tandem affinity purification of ubiquitylated RALB . 6xHis - ubiquitin Flag - RALB - G23V were co - transfected into HEK293T , ubiquitylated RALB was purified using anti - Flag resin followed by Co2 + metal affinity chromatography . Isolated RALB was visualized by immunoblotting using anti - RALB antibody .

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(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 RALB domains ( upper panel ) . RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63 % of RALB acid ( Supplementary Fig . S4a ) . major identified ubiquitylation sites , Lys 47 Lys 160 , are located in effector - interacting domain ( green ) G5 box ( red ) , respectively .

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(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 . (

(c) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB. (


( d ) USP33 regulates RALB ubiquitylation at Lys 47 . ( e ) GTP - GDP status of RALB affects RALB ubiquitylation at Lys 47 . For c - e , 6xHis - tagged ubiquitin indicated RALB mutants were introduced into HEK293T expressing GFP shRNA or USP33 shRNA . Ubiquitylated RALB was purified by Co2 + metal affinity chromatography detected by antibodies specific to RALB . WT , wild type .

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(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 . indicated Flag - tagged RALB mutants were overexpressed in HEK293T then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - SEC5 antibody .

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(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 then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - EXO84 antibody .

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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 . activity of RALB mutants was determined by a RALBP1 - RBD binding .

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(h) Ubiquitylation at Lys 47 does not affect RALB activity. The activity of RALB mutants was determined by a RALBP1-RBD binding assay.


( i ) Rosetta v3 protocol described in ref . was used for ubiquitin - docking simulations . plot represents energies of produced decoys distributed by root - mean - square deviation ( RMSD ) of ubiquitin position relative to RALA from an arbitrarily chosen decoy .

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(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 ubiquitylated - RALA - EXO84 - RBD complexes . Superposition of top ten refined solutions of ubiquitylated - RALA - SEC5 - RBD , selected by combined scores of two independent docking simulations of ubiquitylated - RALA - SEC5 - RBD ubiquitylated - RALA ( left ) . Simulated ubiquitin positions linked to Lys 47 of RALA were combined with RALA - EXO84 ( right ) . Uncropped of blots are shown in Supplementary Fig . S7 .

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(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.


( a ) USP33 does not affect RALB activity . activity of RALB was determined in HEK293T expressing GFP shRNA or USP33 shRNA empty vector ( V ) or HA - tagged USP33 by RALBP1 - RBD binding .

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(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 SEC5 . Flag - tagged RALB was overexpressed in HEK293T expressing GFP shRNA , USP33 shRNA , empty vector ( V ) or HA - tagged USP33 . indicated were then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - Flag or anti - SEC5 antibodies .

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(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 expressing indicated constructs . presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - HA or anti - EXO84 antibody .

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(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 SEC5 . At 24 h after co - transfection with RALB - G23V - EGFP HA - EXO84 , HeLa were immunostained with anti - HA anti - SEC5 antibodies . Scale bars , 10 μm . outlined areas are shown at higher magnification in bottom panels .

(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 SEC5 . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T overexpressing RALB - G23V or RALB - G23V - K47R . presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti - EXO84 antibody .

(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 formation . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T expressing indicated constructs followed by immunoblotting using anti - EXO84 antibody .

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(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 expressing indicated constructs . 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 of USP33 suppression or overexpression . Immunoblot was performed using indicated antibodies . Uncropped of blots are shown in Supplementary Fig . S7 .

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(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.


( a ) Suppression of USP33 facilitates TBK1 - SEC5 formation . Flag - tagged SEC5 was introduced into HEK293T expressing empty vector ( V ) or HA - tagged RALB - G23V together with GFP shRNA or USP33 shRNA as indicated , then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using antibody specific for TBK1 .

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(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 RALB - SEC5 in response to poly ( I : C ) treatment . Flag - tagged SEC5 indicated HA - tagged RALB mutants were introduced into 293 - hTLR3 . At 48 h after transfection were treated 100 μg ml − 1 of poly ( I : C ) for 6 h . Flag - tagged SEC5 was immunoprecipitated with anti - Flag ( M2 ) , analysed for co - precipitation by immunoblotting using anti - HA antibody .

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(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 IRF3 . At 48 h after transfection with empty vector ( V ) , Flag - RALB - G23V Flag - RALB - G23V - K47R , 293 - hTLR3 were treated with 10 μg ml − 1 of poly ( I : C ) for 6 h . Immunoblot was performed using indicated antibodies . Uncropped of blots are shown in Supplementary Fig . S7 .

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

(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.


( a - d ) USP33 inhibits assembly of SEC5 - beclin - 1 complexes . indicated were immunoprecipitated from expressing GFP shRNA or USP33 shRNA with anti - Flag ( M2 ) , anti - HA or anti - SEC5 antibody analysed for co - precipitation by immunoblotting using specific antibodies as indicated .

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(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 interaction between EXO84 beclin 1 . Endogenous beclin 1 was immunoprecipitated from HEK293T , expressing either empty vector ( V ) , HA - USP33 wild type ( WT ) or 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 .

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(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 indicated RAL mutants were overexpressed in HEK293T . At 48 h after transfection were deprived of nutrients incubated in Hank ' s buffered salt solution ( HBSS ) medium for 90 min . Ubiquitylated RALproteins were purified by Co2 + metal affinity chromatography analysed by immunoblotting using anti - RALA or anti - RALB antibodies .

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(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 accumulation of USP33 . HEK TE were incubated in HBSS medium for indicated periods of time . Immunoblotting was performed using anti - USP33 antibody .

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(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 absence of nutrients . At 48 h after co - transfection with RALB - G23V - EGFP USP33 - mCherry , HeLa were incubated in HBSS medium for 90 min . were imaged for EGFP mCherry . Scale bars , 10 μm . outlined areas are shown at higher magnification in bottom panels . Arrows indicate USP33 co - localization to RALB - positive vesicles .

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(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 were transfected with beclin - 1 - HA USP33 - mCherry . At 48 h after transfection , were incubated in HBSS medium for 90 minutes treated as in i . Scale bars , 10 μm . outlined areas are shown at higher magnification in bottom panels . Uncropped of blots are shown in Supplementary Fig . S7 .

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(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.


( a ) USP33 depletion inhibits accumulation of LC3 - lipid conjugates . HEK TE stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h , in presence or absence of 50 μM of chloroquine ( CQ ) . were then assayed for relative accumulation of LC3 - I LC3 - II by immunoblotting using anti - LC3 antibody . LC3 - II / GAPDH ratios have been calculated using ImageJ densitometric .

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(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 affect accumulation of LC3punctae . Wild - type USP33 or a USP33 - shRNA - resistant USP33 mutant ( USP33R ) was introduced into HEK TE stably expressing GFP shRNA or USP33 shRNA . generated were incubated in HBSS medium for 4 h . USP33 was analysed by immunoblotting using anti - USP33 antibody ( b ) . Representative of LC3immunostaining are shown ( c ) . Endogenous LC3punctae were quantified using In Analyser 2000 automatic imaging system IN Investigator software . mean distribution of LC3punctae per is represented as mean of two independent experiments . Statistics source data for d can be found in Supplementary Table S1 ( d ) .

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(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 of p62 . HEK TE expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for indicated periods of time . Immunoblotting was performed using indicated antibodies .

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(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 RALB - G23V - K47R mutant leads to accumulation of LC3 - lipid conjugates . HEK293T overexpressing RALB - G23V or RALB - G23V - K47R mutants were assayed for relative accumulation of LC3 - I LC3 - II by immunoblotting using anti - LC3 antibody .

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(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.


( A ) Western Blot detection of ATM pS1981 , SMCHD1 , TRF2 hnRNPA1 loading control in wild - type two SMCHD1 knockout ( KO1 KO2 ) HeLa transfected with shTRF2 plasmid or EV control .

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(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 SMCHD1 knockout HeLa transfected with shTRF2 plasmid or EV control . Telomeric signals were detected with Cy3 - OO - ( CCCTAA ) 3 are false colored in red , is stained with DAPI is false colored in cyan . Scale bar : 5µm .

(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 ) 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 - Student ' s t - .

(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 cycle profiles from two biological replicates of WT SMCHD1 KO HeLa transfected with shTRF2 plasmid or EV control . were stained with propidium iodide analyzed by Flow cytometry .

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(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.


( A ) Western Blot detection of SMCHD1 , TRF2 hnRNPA1 in SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 treated with or without doxycycline for indicated number of days ( d7 , d8 , d11 ) .

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(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 ) of telomere fusions in SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 treated with or without doxycycline for 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 - Student ' s t - .

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(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 ) of telomeric to detect 3 ' overhang processing of genomic isolated from SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 treated as in experiment in ( B ) . ( Left ) Radiolabeled ( CCCTAA ) n probe was hybridized with a short run ( upper panel ) long run ( lower panel ) native gel to detect signal of telomeric 3 ' overhang . Samples used for short long run were from same digestion split into two . Exo I treatment ( + Exo ) was used as a control that single stranded telomeric signal was terminal . ( Right ) total TTAGGG signal in same lane was deteced upon denaturation hybridization with same probe .

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(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 ) of telomeric overhang signal at d11 after doxycyclin addition to SMCHD1 wild - type SMCHD1 knockout HeLa shTRF2 inducible . bar graph represents average overhang signal from two biological replicates as of signal in untreated with doxycycline with error bars representing standard deviation ( SD ) of sample .

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(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.


A Western blot detection of ATM pS1981 , SMCHD , TRF2 , γH2AX , hnRNPA1 in WT or SMCHD1 KO Hela transfected with shTRF2 EV control .

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A 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 for CO - FISH on metaphase spreads in wild type ( WT ) Hela U2OS . leading strand telomeres were labeled by PNA - FISH with a ( TTAGGG ) 3 - Alexa488 probe ( green ) , lagging strand with a ( CCCTAA ) 3 - Cy5 probe ( pink ) , was stained with DAPI . Examples of telomere sister chromatid exchange ( t - SCEs ) events are marked with arrows . Scale bar :

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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: 5µm


C of chromosome ends with t - SCEs detected by CO - FISH . Data represent means of 2 independent experiments ± SD ( > 700 telomeres / condition / experiment ) with average values indicated .

C 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.


( A - C ) Representative for detection of ATM pS9181 , γH2AX , 53BP1 at telomeres in wild - type ( WT ) SMCHD1 knockout HeLa transfected with shTRF2 plasmid empty vector ( EV ) control . Immunofluoresence ( IF ) for ATM pS1981 ( gray ) , γH2AX ( green ) 53BP1 ( yellow ) was combined with telomeric ( CCCTAA ) 3 - FISH ( red ) DAPI total . Scale bars :

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(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 ) of number of containing > 5 Telomere dysfunction Induced Foci ( TIFs ) detected Data represent mean of 4 independent experiments ± SD ( > 200 / condition / experiment ) for ATM pS1981 3 independent experiments ± SD ( > 200 / condition / experiment ) for γH2AX 53BP1 . ( * * * ) P < 0 . 001 ; unpaired two - Student ' s t - .

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(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 ) of number of 53BP1 TIFs per detected from three independent experiments . Data represent values per with bar representing mean ± SD . ( * * * ) P < 0 . 001 ; unpaired two - Student ' s t - .

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(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.


( A ) Western blot detection of SMCHD1 , ATMpS1981 , CHK2 pT68 , CHK1 pS345 , RPA pS33 , RPA pS4 / 8 , γH2AX Vinculin in wild - type or SMCHD1 KO HeLa γ - irradiated with 3Gy dose harvested at indicated times ( 1h , 12h , 24h ) after treatment .

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(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 for Immunofluoresence ( IF ) detection of ATMpS1981 in wild - type HeLa γ - irradiated with a dose of 3Gy harvested after indicated times . Scale bar :

(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 ) of average number of ATMpS1981 foci per detected as in ( B ) in wild - type SMCHD1 KO γ - irradiated HeLa . Data represent mean of 3 independent experiments ± SD ( > 200 / condition / experiment ) with average values indicated .

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(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.


( A ) Western Blot detection of SMCHD1 , TRF2 , TPP1 hnRNPA1 in wild - type or SMCHD1 knockout HeLa transfected with indicated shRNAs ( shTRF2 , shTPP1 , shTRF2 / shTPP1 ) or EV control .

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(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 transfected with indicated shRNAs or EV control 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 - Student ' s t - .

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(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 transfected with indicated shRNAs or EV control treated for 4 days with ATRi ( VE - 821 ) 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 - Student ' s t - .

(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.


number of neoblasts did not change significantly following inhibition of MTC - gene kiaa1429 . Shown is FISH of smedwi - 1 ( A ) in control kiaa1429 ( RNAi ) ( left ) . smedwi - 1 + were counted following FISH showed no significant difference in expression ( right ; bar represents average normalized ; Materials Methods ) . Scale = 100 µm .

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The 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.


number of neoblasts did not change significantly following inhibition of MTC - gene kiaa1429 . Shown is FISH of h2b ( B ) in control kiaa1429 ( RNAi ) ( left ) . h2b + were counted following FISH showed no significant difference in expression ( right ; bar represents average normalized ; Materials Methods ) . Scale = 100 µm .

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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 isolated from control ( top ) kiaa1429 ( RNAi ) ( bottom ) show an increase in abundance in X2 ( Fincher et al , 2018 ) gate ( green ; Materials Methods ) . ( F ) of in each FACS gate in control kiaa1429 ( RNAi ) showed a six - fold increase in abundance of in X2 gate ( Student ' s t - * * * - p < 0 . 005 . FACS experiments were performed in biological triplicates . Boxes represent IQR , whiskers represent 1 . 5 x IQR , central band represents

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(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


( b ) Coimmuno - precipitation ( IP ) of Fbxo7 - HA Flag - Parkin in whole - lysates from U2OS over - expressing both . IB , immunoblot . Molecular mass markers are in kilodaltons .

(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 transfected with Flag - Parkin or a control ( EGFP ) .

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(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 transfected with Flag - Parkin either full - ( 1 - 522 ) T7 - Fbxo7 or an N - terminal truncation lacking Ubl domain ( 89 - 522 ) .

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(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 expressing Flag - Parkin T7 - Fbxo7 , either full ( 1 - 522 ) or a C - terminal deletion of proline - rich region ( 1 - 398 ) . * indicates IgG heavy chain .

(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 Fbxo7 Ubl domain ( 1 - 88 ) immobilized on glutathione beads . Bead - bound inputs were analyzed by immunoblotting with anti - Parkin antibodies .

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(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 ) disease - causing mutation T22M interferes with Fbxo7 ' s interaction with Parkin . Coimmunoprecipitation was performed as in b using lysates from U2OS expressing Flag - Parkin WT or T22M Fbxo7 - HA .

(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 Fbxo7 in mitochondrial fractions of HEK293T overexpressing both . CxVα GAPDH are markers for mitochondria cytoplasm , respectively . western blots are representative of experiments performed at least three times . Full - blots are presented in Supplementary Figure 9 .

(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.


( a ) Fbxo7 relocates from to mitochondrial fractions of HEK293T treated with CCCP ( 10 μM ) . IB , immunoblot .

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(a) Fbxo7 relocates from the cytosolic to the mitochondrial fractions of HEK293T cells treated with CCCP (10 μM). IB, immunoblot.


( b ) Fbxo7 are increased in Flag - Parkin complexes immunoprecipitated ( IP ) from mitochondrial fraction of HEK293T transfected with Flag - Parkin untagged Fbxo7 following 1 or 3 h treatment with CCCP ( 10 µM ) .

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(b) Fbxo7 levels are increased in Flag-Parkin complexes immunoprecipitated (IP) from the mitochondrial fraction of HEK293T cells transfected with Flag-Parkin and untagged Fbxo7 following 1 or 3 h treatment with CCCP (10 µM).


( c ) Parkin localization at mitochondria was assessed by immunocytochemistry in SH - SY5Y transfected with Flag - Parkin plus scrambled ( scr ) or Fbxo7 siRNA , following 1 or 3 h treatment with CCCP ( 10 μM ) . were scored visually for colocalization of Flag - Parkin with HtrA2 , a mitochondrial marker . are displayed for transfected as indicated , following 0 or 3 h CCCP treatment . For corresponding at 1 h treatment , see Supplementary Figure 2c . Nuclei ( blue ) were stained with DAPI . Scale bars , 10 μm .

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(c) Parkin localization at the mitochondria was assessed by immunocytochemistry in SH-SY5Y cells transfected with Flag-Parkin plus scrambled (scr) or Fbxo7 siRNA, following 1 or 3 h treatment with CCCP (10 μM). Cells were scored visually for the colocalization of Flag-Parkin with HtrA2, a mitochondrial marker. Images are displayed for cells transfected as indicated, following 0 or 3 h CCCP treatment. For corresponding images at 1 h treatment, see Supplementary Figure 2c. Nuclei (blue) were stained with DAPI. Scale bars, 10 μm.


( d ) Loss of Flag - Parkin translocation upon Fbxo7 silencing is rescued by WT R378G Fbxo7 , but not by T22M Fbxo7 , by R498X Fbxo7 or by Fbxo7 in which mitochondrial targeting is mutated ( mt - MTS ) . For c , d , histograms indicate of in which Parkin localized to mitochondria . Data are presented as mean of three experiments ± s . e . m . * P 0 . 05 , * * P 0 . 01 compared to transfected with Flag - Parkin plus Fbxo7 siRNA .

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(d) Loss of Flag-Parkin translocation upon Fbxo7 silencing is rescued by WT and R378G Fbxo7, but not by T22M Fbxo7, by R498X Fbxo7 or by Fbxo7 in which the mitochondrial targeting sequence is mutated (mt-MTS). For c,d, histograms indicate the percentage of cells in which Parkin localized to the mitochondria. Data are presented as mean of three experiments ± s.e.m. *P 0.05, **P 0.01 compared to cells transfected with Flag-Parkin plus Fbxo7 siRNA.


( e ) T7 - tagged WT Fbxo7 localizes to both ( C ) mitochondrial ( M ) fractions of transfected HEK293T , but MTS mutant ( mt - MTS ) Fbxo7 localizes only to fraction . CxVα PDHE1α are mitochondrial markers ; GAPDH is a marker . western blots are representative of experiments performed at least three times . Full - blots are presented in Supplementary Figure 9 .

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(e) T7-tagged WT Fbxo7 localizes to both cytosolic (C) and mitochondrial (M) fractions of transfected HEK293T cells, but MTS mutant (mt-MTS) Fbxo7 localizes only to the cytosolic fraction. CxVα and PDHE1α are mitochondrial markers; GAPDH is a cytosolic marker. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.


( a , b ) Overexpression of Fbxo7 by da - GAL4 ( da > Fbxo7 ) suppresses climbing ( a ) flight ( b ) defects of parkin mutants .

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(a,b) Overexpression of Fbxo7 by da-GAL4 (da>Fbxo7) suppresses climbing (a) and flight (b) defects of parkin mutants.


( c ) Overexpression of Fbxo7 also suppresses dopaminergic ( DA ) neurodegeneration parkin mutants .

(c) Overexpression of Fbxo7 also suppresses dopaminergic (DA) neurodegeneration parkin mutants.


( d - f ) Top middle panels , toluidine blue - stained sections of adult thorax . Bottom panels , transmission electron of muscle . Fbxo7 overexpression suppresses muscle degeneration mitochondrial disruption in parkin mutants . Scale bars : 200 μm ( top ) , 20 μm ( middle ) , 2 μm ( bottom ) . are representative of three per genotype .

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(d-f) Top and middle panels, toluidine blue-stained sections of adult thorax. Bottom panels, transmission electron micrographs of muscle. Fbxo7 overexpression suppresses muscle degeneration and mitochondrial disruption in parkin mutants. Scale bars: 200 μm (top), 20 μm (middle), 2 μm (bottom). Images are representative of three animals per genotype.


( g , h ) Overexpression of Fbxo7 pathogenic mutants , mt - MTS or isoform 2 by da - GAL4 fails to rescue climbing ( g ) flight ( h ) deficits in parkin mutants . Control genotype is park25 / + ; da - GAL4 / + . transgenic lines are site - directed integration except WT ( 4 ) , which is random integration 4 ( see Supplementary Fig . 3 Online Methods ) . Histograms indicate mean ± s . e . m . One - way ANOVA with Bonferroni correction ( * * * P 0 . 001 , * * P 0 . 01 ) . For climbing flight assays , at least 50 flies were assessed ; number of flies is shown for each bar .

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(g,h) Overexpression of Fbxo7 pathogenic mutants, mt-MTS or isoform 2 by da-GAL4 fails to rescue climbing (g) and flight (h) deficits in parkin mutants. Control genotype is park25/+; da-GAL4/+. All transgenic lines are site-directed integration except WT(4), which is random integration line 4 (see Supplementary Fig. 3 and Online Methods). Histograms indicate mean ± s.e.m. One-way ANOVA with Bonferroni correction (***P 0.001, **P 0.01). For climbing and flight assays, at least 50 flies were assessed; number of flies is shown for each bar.


( a ) Coimmunoprecipitation ( IP ) of PINK1 - Myc Flag - Fbxo7 in whole - lysates from U2OS overexpressing both . IB , immunoblot .

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(a) Coimmunoprecipitation (IP) of PINK1-Myc and Flag-Fbxo7 in whole-cell lysates from U2OS cells overexpressing both proteins. IB, immunoblot.


( b ) Coimmunoprecipitation of PINK1 - Myc with full - two N - terminally deleted Flag - Fbxo7 forms . PINK1 - Myc is detected at low in with Flag - Fbxo7 ( 89 - 522 ) but not with Flag - Fbxo7 ( 129 - 522 ) .

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(b) Coimmunoprecipitation of PINK1-Myc with full-length and two N-terminally deleted Flag-Fbxo7 forms. PINK1-Myc is detected at low levels in complex with Flag-Fbxo7(89-522) but not with Flag-Fbxo7(129-522).


( c ) As in b , using lysates from U2OS expressing PINK1 - Myc , Flag - Fbxo7 containing either N - or C - terminal truncations .

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(c) As in b, using lysates from U2OS cells expressing PINK1-Myc, and Flag-Fbxo7 containing either N- or C-terminal truncations.


( d ) In vitro coprecipitation experiments were performed using in vitro - translated ( IVT ) PINK1 - Myc either GST or GST fusions of Fbxo7 ( 1 - 398 ) or Fbxo7 ( 129 - 398 ) immobilized on glutathione beads .

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(d) In vitro coprecipitation experiments were performed using in vitro-translated (IVT) PINK1-Myc and either GST or GST fusions of Fbxo7(1-398) or Fbxo7(129-398) immobilized on glutathione beads.


( e ) Competitive binding assays using immobilized GST - Fbxo7 ( 1 - 398 ) incubated with IVT Flag - Parkin / or full ( top panel ) or N - terminally truncated ( bottom panel ) PINK1 - Myc . Input bead - bound were analyzed by immunoblotting as indicated .

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(e) Competitive binding assays using immobilized GST-Fbxo7(1-398) incubated with IVT Flag-Parkin and/or full length (top panel) or N-terminally truncated (bottom panel) PINK1-Myc. Input and bead-bound proteins were analyzed by immunoblotting as indicated.


( f ) As in e , immobilized GST - ΔN - PINK1 ( top panel ) , GST - Parkin ( middle panel ) immobilized GST alone ( bottom panel ) were incubated with combinations of IVT Flag - Parkin , T7 - ΔN - PINK1 Fbxo7 - HA as indicated . Input bead - bound were analyzed by immunoblotting with indicated antibodies . western blots were performed a minimum of three times . Full - blots are presented in Supplementary Figure 9 .

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(f) As in e, immobilized GST-ΔN-PINK1 (top panel), GST-Parkin (middle panel) and immobilized GST alone (bottom panel) were incubated with combinations of IVT Flag-Parkin, T7-ΔN-PINK1 and Fbxo7-HA as indicated. Input and bead-bound proteins were analyzed by immunoblotting with the indicated antibodies. All western blots were performed a minimum of three times. Full-length blots are presented in Supplementary Figure 9.