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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 ) .

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

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 .

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

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 - 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 .

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

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 - 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 .

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

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 cell lineages with different fates . Vertical bars represent fresh 7H9 medium ( white ) , CIP ( yellow ) , and 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

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 .

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

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 .

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

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 .

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

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 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 ) .

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

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 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 ) .

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

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 ) .

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

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 . 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 ) .

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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 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 ±

O O O O O O O O O O O O O O O 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 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 the red line .

O O O O O O 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 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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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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 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 .

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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 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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 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 .

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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 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 .

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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 . (

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(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 .

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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 . 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 .

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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 cells and 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 . The activity of RALB mutants was determined by a RALBP1 - RBD binding assay .

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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 ) 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 .

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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 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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(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 . 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 .

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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 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 .

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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 cells expressing the indicated constructs . The 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 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 .

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(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 .

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(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 .

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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 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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(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 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 .

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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 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 .

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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.


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( 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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(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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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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(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 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 .

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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 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 ) .

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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 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 .

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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 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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(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 and hnRNPA1 loading control in wild - type and two SMCHD1 knockout ( KO1 and KO2 ) HeLa cells 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 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 .

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(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 .

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(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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(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 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 ) .

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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 ) 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 .

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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 ) 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 .

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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 ) 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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(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 , and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and 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 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 :

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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 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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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 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 :

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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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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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(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 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 .

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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 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 :

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(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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(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 and hnRNPA1 in wild - type or SMCHD1 knockout HeLa cells transfected with the 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 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 .

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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 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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(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.


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 .

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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.


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 .

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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 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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(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 and Flag - Parkin in whole - cell lysates from U2OS cells over - expressing both proteins . IB , immunoblot . Molecular mass markers are in kilodaltons .

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(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 ) .

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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 cells transfected with Flag - Parkin and either full - length ( 1 - 522 ) T7 - Fbxo7 or an N - terminal truncation lacking the 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 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 .

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(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 .

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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 ) 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 .

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(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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(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 the cytosolic to the mitochondrial fractions of HEK293T cells 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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) and 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 .

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


( 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 .

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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 ) 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 .

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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 and Flag - Fbxo7 in whole - cell lysates from U2OS cells overexpressing both proteins . 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 - 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 ) .

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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 cells expressing PINK1 - Myc , and 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 and 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 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 .

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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 ) 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 .

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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.


( a ) PINK1 localization at the mitochondria was assessed by immunocytochemistry in SH - SY5Y cells transfected with PINK1 - HA plus scrambled ( scr ) or Fbxo7 siRNA following 1 or 3 h treatment with CCCP ( 10 μM ) . Cells were scored visually for the colocalization of PINK1 - HA with complex Vβ subunit ( CxVβ ) , a mitochondrial marker . Nuclei ( blue ) are stained with DAPI . Histograms indicate the percentage of cells in which PINK1 - HA accumulated at the mitochondria . Data are presented as mean ± s . e . m . ; * P 0 . 05 . Representative images are displayed for cells transfected as indicated , following 0 or 3 h CCCP treatment . For corresponding images at 0 h and 1 h treatment , see Supplementary Figure 5a . Scale bars , 10 μm .

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(a) PINK1 localization at the mitochondria was assessed by immunocytochemistry in SH-SY5Y cells transfected with PINK1-HA plus scrambled (scr) or Fbxo7 siRNA following 1 or 3 h treatment with CCCP (10 μM). Cells were scored visually for the colocalization of PINK1-HA with complex Vβ subunit (CxVβ), a mitochondrial marker. Nuclei (blue) are stained with DAPI. Histograms indicate the percentage of cells in which PINK1-HA accumulated at the mitochondria. Data are presented as mean ± s.e.m.; *P 0.05. Representative images are displayed for cells transfected as indicated, following 0 or 3 h CCCP treatment. For corresponding images at 0 h and 1 h treatment, see Supplementary Figure 5a. Scale bars, 10 μm.


( b ) Fbxo7 accumulation in the mitochondrial fraction following treatment with CCCP ( 10 μM ) is impaired in SH - SY5Y cells transfected with PINK1 siRNA compared to scrambled siRNA ( scr ) . IB , immunoblot . Full - length blots are presented in Supplementary Figure 9 .

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(b) Fbxo7 accumulation in the mitochondrial fraction following treatment with CCCP (10 μM) is impaired in SH-SY5Y cells transfected with PINK1 siRNA compared to scrambled siRNA (scr). IB, immunoblot. Full-length blots are presented in Supplementary Figure 9.


( c - f ) Overexpression of Fbxo7 does not rescue climbing ( c , e ) or flight ( d , f ) defects in PINK1 male mutants ( PINK1B9 ) or PINK1 : parkin double mutants ( PINK1B9 ; park25 , daG4 ) . Control genotypes are PINK1B9 / + ; da - GAL4 / + ( c , d ) and da - GAL4 / + ( e , f ) . Histograms indicate mean ± s . e . m . One - way ANOVA with Bonferroni correction ( * * * P 0 . 001 ) . For climbing and flight assays , at least 50 flies were assessed ; number of flies is shown for each bar for c - f .

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(c-f) Overexpression of Fbxo7 does not rescue climbing (c,e) or flight (d,f) defects in PINK1 male mutants (PINK1B9) or PINK1:parkin double mutants (PINK1B9;park25, daG4). Control genotypes are PINK1B9/+; da-GAL4/+ (c,d) and da-GAL4/+ (e,f). Histograms indicate mean ± s.e.m. One-way ANOVA with Bonferroni correction (***P 0.001). For climbing and flight assays, at least 50 flies were assessed; number of flies is shown for each bar for c-f.


( a , b ) Ubiquitination of Mfn1 following treatment with CCCP ( 10 μM ) is reduced in the mitochondrial fraction of both SH - SY5Y cells stably expressing Fbxo7 short hairpin RNA ( Fbxo7 KD ) compared to an empty vector control line ( a ) and in patient fibroblasts with homozygous R378G mutation compared to fibroblasts from healthy controls ( b ) . Arrowheads indicate ubiquitinated Mfn1 . IB , immunoblot .

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(a,b) Ubiquitination of Mfn1 following treatment with CCCP (10 μM) is reduced in the mitochondrial fraction of both SH-SY5Y cells stably expressing Fbxo7 short hairpin RNA (Fbxo7 KD) compared to an empty vector control line (a) and in patient fibroblasts with homozygous R378G mutation compared to fibroblasts from healthy controls (b). Arrowheads indicate ubiquitinated Mfn1. IB, immunoblot.


( c , d ) Fbxo7 expression restores elevated Mfn steady - state levels in parkin ( c ) but not PINK1 ( d ) mutant Drosophila . Histograms show mean ± s . e . m . of densitometry analysis of Mfn immunoblots above , normalized to complex Vα ( CxVα ) . Control genotypes are park25 / + ; da - GAL4 / + ( c ) and PINK1B9 / + ; da - GAL4 / + ( d ) .

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(c,d) Fbxo7 expression restores elevated Mfn steady-state levels in parkin (c) but not PINK1 (d) mutant Drosophila. Histograms show mean ± s.e.m. of densitometry analysis of Mfn immunoblots above, normalized to complex Vα (CxVα). Control genotypes are park25/+; da-GAL4/+ (c) and PINK1B9/+; da-GAL4/+ (d).


( e ) Mitochondria in control Drosophila S2R + cells stained with MitoTracker Red show a heterogeneous morphology , with a mixture of tubules and fragmented mitochondria . RNAi knockdown of parkin or PINK1 causes excessive fusion and elongated mitochondria compared to control double - stranded RNA ( Caenorhabditis elegans gene ZK686 . 3 ) . Expression of Fbxo7 restores parkin but not PINK1 knockdown phenotype to WT appearance . Scale bar shows 5 μm . ( f ) Quantification of mitochondrial morphology in dsRNA treated cells . Scoring system : 1 , fragmented ; 2 , WT ; 3 , tubular ; 4 , hyper - fused ( clumped ) . Histograms indicate mean ± s . e . m . Two - tailed Student t - tests ( * * * P 0 . 001 , * P 0 . 05 ) . All western blots were performed a minimum of three times and images are representative of 100 cells scored per condition . Full - length blots are presented in Supplementary Figure 9 .

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(e) Mitochondria in control Drosophila S2R+ cells stained with MitoTracker Red show a heterogeneous morphology, with a mixture of tubules and fragmented mitochondria. RNAi knockdown of parkin or PINK1 causes excessive fusion and elongated mitochondria compared to control double-stranded RNA (Caenorhabditis elegans gene ZK686.3). Expression of Fbxo7 restores parkin but not PINK1 knockdown phenotype to WT appearance. Scale bar shows 5 μm. (f) Quantification of mitochondrial morphology in dsRNA treated cells. Scoring system: 1, fragmented; 2, WT; 3, tubular; 4, hyper-fused (clumped). Histograms indicate mean ± s.e.m. Two-tailed Student t-tests (***P 0.001, *P 0.05). All western blots were performed a minimum of three times and images are representative of 100 cells scored per condition. Full-length blots are presented in Supplementary Figure 9.


( a ) Treatment with CCCP ( 10 μM ) results in an increase in LC3 - II in the mitochondrial but not the cytosolic fraction of cells stably expressing the empty shRNA vector ( control ) , and this is delayed in stable Fbxo7 knockdown ( KD ) SH - SY5Y cells .

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(a) Treatment with CCCP (10 μM) results in an increase in LC3-II in the mitochondrial but not the cytosolic fraction of cells stably expressing the empty shRNA vector (control), and this is delayed in stable Fbxo7 knockdown (KD) SH-SY5Y cells.


( b ) As in a , an accumulation of LC3 - II was observed in the mitochondrial fraction of healthy control fibroblasts following 1 and 5 h CCCP treatment , but this was reduced in fibroblasts from a patient carrying the R378G mutation . Western blots were performed a minimum of three times .

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(b) As in a, an accumulation of LC3-II was observed in the mitochondrial fraction of healthy control fibroblasts following 1 and 5 h CCCP treatment, but this was reduced in fibroblasts from a patient carrying the R378G mutation. Western blots were performed a minimum of three times.


( c ) Mitochondrial accumulation of p62 following CCCP treatment is inhibited by Fbxo7 siRNA . Flag - Parkin overexpressing SH - SY5Y cells were transfected with scrambled ( scr ) or Fbxo7 siRNA as indicated and treated with either DMSO or CCCP ( 10 μM ) for 6 h . Colocalization of p62 with HtrA2 , a mitochondrial marker , was assessed by Pearson ' s correlation coefficient ( Rr ) on a cell - by - cell basis . Histogram shows the percentage of cells in which Rr was greater than 0 . 5 . Data are represented as mean ± s . e . m . , * P 0 . 05 . Scale bars , 10 μm .

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(c) Mitochondrial accumulation of p62 following CCCP treatment is inhibited by Fbxo7 siRNA. Flag-Parkin overexpressing SH-SY5Y cells were transfected with scrambled (scr) or Fbxo7 siRNA as indicated and treated with either DMSO or CCCP (10 μM) for 6 h. Colocalization of p62 with HtrA2, a mitochondrial marker, was assessed by Pearson's correlation coefficient (Rr) on a cell-by-cell basis. Histogram shows the percentage of cells in which Rr was greater than 0.5. Data are represented as mean ± s.e.m., *P 0.05. Scale bars, 10 μm.