( 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 .

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

(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 .

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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 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 .

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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 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 .

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

(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 .

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

(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 .

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

(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 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 .

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

(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 .

O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O

(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 .

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

(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 ) .

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

(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 .

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

(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 .

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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 ) 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 - .

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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 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 .

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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 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 :

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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 ) 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 - .

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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.


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

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 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

(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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 .

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-CONTROLLED_VAR O O O O O O O O O O O O O O

(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 .

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

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

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

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

O O 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 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

(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 mutants ( PINK1B9 ) or PINK1 : parkin double mutants ( PINK1B9 ; park25 , daG4 ) . Control genotypes are PINK1B9 / + ; da - GAL4 / + ( c , d ) da - GAL4 / + ( e , f ) . Histograms indicate mean ± s . e . m . One - way ANOVA with Bonferroni correction ( * * * P 0 . 001 ) . For climbing flight assays , at least 50 flies were assessed ; number of flies is shown for each bar for c - f .

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

(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 mitochondrial fraction of both SH - SY5Y stably expressing Fbxo7 short hairpin ( Fbxo7 KD ) compared to an empty vector control ( a ) in fibroblasts with homozygous R378G mutation compared to fibroblasts from healthy controls ( b ) . Arrowheads indicate ubiquitinated Mfn1 . IB , immunoblot .

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

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

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

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

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

(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 mitochondrial but not fraction of stably expressing empty shRNA vector ( control ) , this is delayed in stable Fbxo7 knockdown ( KD ) SH - SY5Y .

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

(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 mitochondrial fraction of healthy control fibroblasts following 1 5 h CCCP treatment , but this was reduced in fibroblasts from a carrying R378G mutation . Western blots were performed a minimum of three times .

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 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

(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 were transfected with scrambled ( scr ) or Fbxo7 siRNA as indicated 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 - by - basis . Histogram shows of in which Rr was greater than 0 . 5 . Data are represented as mean ± s . e . m . , * P 0 . 05 . Scale bars , 10 μm .

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

(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.


( d ) Mitophagy was analyzed in untransfected ( UT ) SH - SY5Y or in stable Flag - Parkin overexpressing SH - SY5Y transfected with either scrambled ( scr ) or Fbxo7 siRNA . Histogram indicates of with no remaining mitochondria following 24 h treatment with CCCP ( 10 μM ) for each condition . Vβ subunit ( CxVβ ) was used as a mitochondrial marker . Data are presented as mean ± s . e . m . , * * P 0 . 01 . Scale bars , 10 μm .

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 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 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

(d) Mitophagy was analyzed in untransfected (UT) SH-SY5Y cells or in stable Flag-Parkin overexpressing SH-SY5Y cells transfected with either scrambled (scr) or Fbxo7 siRNA. Histogram indicates the percentage of cells with no remaining mitochondria following 24 h treatment with CCCP (10 μM) for each condition. Complex Vβ subunit (CxVβ) was used as a mitochondrial marker. Data are presented as mean ± s.e.m., **P 0.01. Scale bars, 10 μm.


( e ) Mitochondrial mass was measured in Flag - Parkin overexpressing SH - SY5Y transfected with scrambled ( scr ) or Fbxo7 siRNA treated for 24 h with either dimethylsulfoxide ( DMSO ) vehicle or CCCP ( 10 μM ) . For representative , see Supplementary Figure 8d . Full - blots are presented in Supplementary Figure 9 .

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 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

(e) Mitochondrial mass was measured in Flag-Parkin overexpressing SH-SY5Y cells transfected with scrambled (scr) or Fbxo7 siRNA and treated for 24 h with either dimethylsulfoxide (DMSO) vehicle or CCCP (10 μM). For representative images, see Supplementary Figure 8d. Full-length blots are presented in Supplementary Figure 9.


HUVECs transfected with control or CEP41 siRNAs were scratched ( 0 h ) to induce wounding then incubated for 12 h to allow wound closure . wound margins were observed every 4 h in CEP41 # 1 # 2 siRNA - transfected compared to those of control . Representative of subjected to wound closure in ( A ) . Scale bars , 600 μm . of extent of wound closure in ( B ) presented graphically by measuring between dotted lines at each time point .

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 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

HUVECs transfected with control or CEP41 siRNAs were scratched (0 h) to induce wounding and then incubated for 12 h to allow wound closure. The wound margins were observed every 4 h in the CEP41#1 and #2 siRNA-transfected cells and compared to those of control cells. Representative images of cells subjected to the wound closure assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (B) presented graphically by measuring the distance between the dotted lines at each time point.


siRNA - transfected HUVECs were plated inside a transwell chamber incubated with serum for 18 h . that invaded were observed after with crystal violet ( CV ) solution . Scale bars , 600 μm . numbers of that invaded in each field of view was counted with ImageJ software in ( 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

The siRNA-transfected HUVECs were plated inside a transwell chamber and incubated with serum for 18 h. The cells that invaded were observed after staining with crystal violet (CV) solution. Scale bars, 600 μm. The numbers of cells that invaded in each field of view was counted with the ImageJ software in (D).


Tubulogenesis of control CEP41 - knockdown for 18 h was compared via an in vitro angiogenesis . Scale bars , 600 μm . of tube node numbers in ( F ) tube in ( G ) from each field of view using ImageJ angiogenesis analyzer at indicated time points . graph compares relative of control CEP41 - knockdown . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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 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

Tubulogenesis of control and CEP41-knockdown cells for 18 h was compared via an in vitro angiogenesis assay. Scale bars, 600 μm. Quantification of tube node numbers in (F) and tube length in (G) from each field of view using the ImageJ angiogenesis analyzer at the indicated time points. The graph compares the relative length of control cells and CEP41-knockdown cells. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


Blood vessels were observed in MOs ( control , cep41 AUG ( 2 . 5 ng ) , or cep41 SB ( 2 ng ) ) - injected or cep41 - mutated Tg ( kdrl : eGFP ) zebrafish at 40 hours post - fertilization ( hpf ) by microscopy . Asterisks arrowheads indicate impaired ISVs DLAVs , respectively . representative for of ISV lumen diameter are indicated by dotted rectangles . A , anterior ; P , posterior ; DLAV , dorsal longitudinal anastomotic vessel ; ISV , intersegmental vessel . Scale bars , 100 μm . of ISV lumen diameter in ( B ) , numbers of defective ISVs in ( C ) , numbers of with aberrant DLAVs in ( D ) , numbers of ruptured DLAVs in ( E ) from data observed in equivalent fields of view ( within eight somites ) . severity of blood vessel defects in cep41 - deficient zebrafish : B ( narrowed ISVs ) < C ( shorten , fused , missing ISVs ) < D E ( ruptured DLAVs ) . Data are shown as mean ± SD of three independent experiments with ≥ 20 per condition .

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 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 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

Blood vessels were observed in MOs (control, cep41 AUG (2.5 ng), or cep41 SB (2 ng))-injected or cep41-mutated Tg(kdrl:eGFP) zebrafish at 40 hours post-fertilization (hpf) by fluorescent microscopy. Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. The representative images for analysis of ISV lumen diameter are indicated by dotted rectangles. A, anterior; P, posterior; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel. Scale bars, 100 μm. Quantification of ISV lumen diameter in (B), the numbers of defective ISVs in (C), the numbers of embryos with aberrant DLAVs in (D), and the numbers of ruptured DLAVs in (E) from data observed in equivalent fields of view (within eight somites). The severity of blood vessel defects in cep41-deficient zebrafish: B (narrowed ISVs) < C (shorten, fused, and missing ISVs) < D and E (ruptured DLAVs). Data are shown as mean ± SD of three independent experiments with ≥ 20 embryos per condition.


CV protrusions ( arrowheads ) vascular loops ( asterisks ) were observed in zebrafish at 35 hpf . within dotted rectangles are magnified in right panels . A , anterior ; P , posterior ; CA , caudal artery ; CV , caudal vein . Scale bars , 40 μm . of of CV protrusions in ( G ) presented graphically by measuring them in equivalent fields of view ( within five somites ) .

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

CV protrusions (arrowheads) and vascular loops (asterisks) were observed in zebrafish at 35 hpf. The images within the dotted rectangles are magnified in the right panels. A, anterior; P, posterior; CA, caudal artery; CV, caudal vein. Scale bars, 40 μm. Quantification of the length of the CV protrusions in (G) presented graphically by measuring them in equivalent fields of view (within five somites).


Control or CEP41 siRNA - transfected HUVECs were immunostained with Ac - Tub - or GT335 - specific antibodies in ( A ) . rectangles indicate representative from each immunostaining experiment presented as magnified in right panels . Scale bars , 20 μm . of ciliated numbers in in ( A ) from results of three independent experiments with ≥ 200 per condition ( mean ± SD ) in ( B ) .

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

Control or CEP41 siRNA-transfected HUVECs were immunostained with Ac-Tub- or GT335-specific antibodies in (A). The rectangles indicate the representative cells from each immunostaining experiment presented as magnified images in the right panels. Scale bars, 20 μm. Quantification of the ciliated cell numbers in the images in (A) from the results of three independent experiments with ≥ 200 cells per condition (mean ± SD) in (B).


Wild - type cep41 - deficient Tg ( kdrl : eGFP ) zebrafish were fixed for immunostaining with Ac - Tub - or GT335 - specific antibodies at 28 hpf in ( C ) . rectangles indicate arterial cilia ( AC ) venous cilia ( VC ) in zebrafish endothelial ( EC ) . Magnified representative are displayed in right panels . Scale bars , 40 μm . of labeled cilia observed in equivalent fields of view are presented graphically in ( D ) . Data are shown as median of three independent experiments ( n ≥ 20 per condition ) .

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

Wild-type and cep41-deficient Tg(kdrl:eGFP) zebrafish embryos were fixed for immunostaining with Ac-Tub- or GT335-specific antibodies at 28 hpf in (C). The rectangles indicate the arterial cilia (AC) and venous cilia (VC) in zebrafish endothelial cells (EC). Magnified representative images are displayed in the right panels. Scale bars, 40 μm. Quantification of the labeled cilia observed in equivalent fields of view are presented graphically in (D). Data are shown as median of three independent experiments (n ≥ 20 embryos per condition).


HUVECs transfected with CCP5 / or CEP41 siRNAs were scratched incubated to evaluate wound closure . extent of wound closure in co - transfected with CCP5 CEP41 siRNAs was compared with that in control or CEP41 siRNA - transfected at indicated time points ( A ) . Scale bars , 600 μm . of extent of wound closure in ( A ) is presented graphically by measuring between dotted lines at each time point in ( B ) . Data are shown as mean ± SD of three independent experiments ( n ≥ 3 scratches per experimental condition ) .

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

HUVECs transfected with CCP5 and/or CEP41 siRNAs were scratched and incubated to evaluate wound closure. The extent of wound closure in co-transfected cells with CCP5 and CEP41 siRNAs was compared with that in control or CEP41 siRNA-transfected cells at the indicated time points (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) is presented graphically by measuring the distance between the dotted lines at each time point in (B). Data are shown as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition).


indicated siRNA - transfected HUVECs were subjected to in vitro angiogenesis assays . Tubulogenesis of HUVECs depleted for CEP41 CCP5 was compared to that of control , CEP41 single - knockdown , or CCP5 single - knockdown ( C ) . Scale bars , 600 μm . of tube node number in ( D ) tube in ( E ) from data examined within equivalent fields of view at indicated time points using ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

The indicated siRNA-transfected HUVECs were subjected to in vitro angiogenesis assays. Tubulogenesis of HUVECs depleted for CEP41 and CCP5 was compared to that of control, CEP41 single-knockdown, or CCP5 single-knockdown cells (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at the indicated time points using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


blood vessels in trunks of cep41 - mutant / morphant zebrafish were compared to those of zebrafish co - injected with ccp5 MOs ( 2 ng ) at 40 hpf in ( F ) . Asterisks arrowheads indicate impaired ISVs DLAVs , respectively . A , anterior ; P , posterior . Scale bars , 100 μm . of number of defective ISVs DLAVs from data observed in equivalent fields of view ( within eight somites ) . Data are median of three independent experiments with ≥ 20 per condition .

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

The blood vessels in the trunks of cep41-mutant/morphant zebrafish were compared to those of zebrafish co-injected with ccp5 MOs (2 ng) at 40 hpf in (F). Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. A, anterior; P, posterior. Scale bars, 100 μm. Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view (within eight somites). Data are median of three independent experiments with ≥ 20 embryos per condition.


HUVECs co - transfected with CEP41 CCP5 siRNAs were immunostained with ARL13b - GT335 - specific antibodies . results were compared to those of control , single CEP41 , or CCP5 siRNA - transfected . Representative indicated with rectangles appear in magnified in lower panels . Scale bars , 20 μm . of relative of GT335 / ARL13b signals in cilia ciliary ( J ) in ( H ) are result of three independent experiments with ≥ 200 per condition ( median ) . * *

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

HUVECs co-transfected with CEP41 and CCP5 siRNAs were immunostained with ARL13b- and GT335-specific antibodies. The staining results were compared to those of control, single CEP41, or CCP5 siRNA-transfected cells. Representative images indicated with rectangles appear in magnified images in the lower panels. Scale bars, 20 μm. Quantification of relative intensity of GT335/ARL13b signals in the cilia ciliary length (J) in images (H) are the result of three independent experiments with ≥ 200 cells per condition (median). **


Control or CEP41 siRNA - transfected HUVECs cultivated under either static or shear stress states examined in a wound healing in ( A ) . Scale bars , 600 μm . of extent of wound closure in ( A ) displayed graphically by measuring between lines at indicated time points in ( B ) . Data are presented as mean ± SD of three independent experiments ( n ≥ 3 scratches per experimental condition ) . Statistical significance was determined using two - way ANOVA followed by Tukey ' s post hoc ( * * * P < 0 . 01 , ns : non - significant ) .

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Control or CEP41 siRNA-transfected HUVECs cultivated under either static or shear stress states and examined in a wound healing assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) displayed graphically by measuring the distance between the lines at the indicated time points in (B). Data are presented as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition). Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (***P < 0.01, ns: non-significant).


in vitro angiogenesis was performed in static or shear stress - exposed HUVECs transfected with indicated siRNAs for 18 h in ( C ) .

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in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C).


in vitro angiogenesis was performed in static or shear stress - exposed HUVECs transfected with indicated siRNAs for 18 h in ( C ) . Scale bars , 600 μm . of tube node number in ( D ) tube in ( E ) from data examined within equivalent fields of view at each time point using ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


Control or CEP41 - deficient HUVECs cultivated under laminar shear stress immunostained with ARL13b - specific antibodies to measure deciliation . Representative are indicated by rectangles with numbers shown in magnified in right panels . resulting data were compared to those of under static conditions in ( F ) . Scale bars , 20 μm . of ARL13b - positive ciliary ( G ) of relative of GT335 / ARL13b signals in cilia ( H ) in ( F ) is results of three independent experiments with ≥ 200 per condition ( mean ± SD ( G ) , median ( H ) ) . *

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

Control or CEP41-deficient HUVECs cultivated under laminar shear stress and immunostained with ARL13b-specific antibodies to measure deciliation. Representative images are indicated by rectangles with numbers and shown in magnified images in the right panels. The resulting data were compared to those of cells under static conditions in (F). Scale bars, 20 μm. Quantification of ARL13b-positive ciliary cells (G) and of relative intensity of GT335/ARL13b signals in the cilia (H) in images (F) is the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (G), median (H)). *


Control , CEP41 - , or CCP5 - depleted hTERT - RPE1 cultured under serum starvation for 48 h fixed at 18 h after serum retrieval for immunostaining with both ARL13b - GT335 - specific antibodies in ( I ) . of results 18 h after serum addition . Representative are indicated by rectangles . Scale bars , 20 μm . of ARL13b - positive ciliated ( J ) relative of GT335 / ARL13b signals in cilia ( K ) under either serum starvation or serum retrieval ( 18 h ) conditions are results of three independent experiments with ≥ 200 per condition ( mean ± SD ( J ) , median ( K ) ) .

O O B-CONTROLLED_VAR 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 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 B-MEASURED_VAR 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

Control, CEP41-, or CCP5-depleted hTERT-RPE1 cells cultured under serum starvation for 48 h and fixed at 18 h after serum retrieval for immunostaining with both ARL13b- and GT335-specific antibodies in (I). Images of the results 18 h after serum addition. Representative cells are indicated by rectangles. Scale bars, 20 μm. Quantification of ARL13b-positive ciliated cells (J) and relative intensity of GT335/ARL13b signals in the cilia (K) under either serum starvation or serum retrieval (18 h) conditions are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (J), median (K)).


VEGFA ( A ) VEGFR2 ( B ) mRNA quantified by qRT - PCR in control or CEP41 - knockdown HUVECs under static or shear stress conditions . expression of GAPDH was quantified for normalization of qRT - PCR results . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined with Brown - Forsythe ANOVA followed by Dunnett ' s T3 post hoc ( * * P < 0 . 01 , * * * P < 0 . 001 , ns : non - significant ) .

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VEGFA (A) and VEGFR2 (B) mRNA levels quantified by qRT-PCR in control or CEP41-knockdown HUVECs under static or shear stress conditions. The expression of GAPDH was quantified for the normalization of the qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined with the Brown-Forsythe ANOVA followed by Dunnett's T3 post hoc test (**P < 0.01, ***P < 0.001, ns: non-significant).


Immunoblot assays for phospho - AURKA AURKA were performed in HUVECs transfected with indicated siRNAs . were normalized against β - ACTIN in same blots .

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Immunoblot assays for phospho-AURKA and AURKA were performed in HUVECs transfected with the indicated siRNAs. Protein levels were normalized against β-ACTIN in the same blots.


Immunoblot assays for phospho - AURKA AURKA were performed in hTERT - RPE1 ( D ) transfected with indicated siRNAs . were normalized against β - ACTIN in same blots .

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Immunoblot assays for phospho-AURKA and AURKA were performed in hTERT-RPE1 cells (D) transfected with the indicated siRNAs. Protein levels were normalized against β-ACTIN in the same blots.


mRNA of zebrafish vegfa ( E ) vegfr2 ( F ) were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish at 18 hpf ( low shear stress ) 26 hpf ( high shear stress ) . expression of vegfa vegfr2 was quantified in neuronal from control MO - injected zebrafish for comparisons with that of ECs . expression of zebrafish β - actin was quantified for normalization of those qRT - PCR results . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined using one - way ANOVA followed by Tukey ' s post hoc ( * * * P < 0 . 001 , ns : non - significant ) .

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 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 B-MEASURED_VAR I-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

The mRNA levels of zebrafish vegfa (E) and vegfr2 (F) were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish at 18 hpf (low shear stress) and 26 hpf (high shear stress). The expression of vegfa and vegfr2 was quantified in neuronal cells from control MO-injected zebrafish for comparisons with that of ECs. The expression of zebrafish β-actin was quantified for the normalization of those qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).


Tg ( kdrl : eGFP ) zebrafish were injected with control - or cep41 - MOs subjected to immunostaining with phospho - AURKA - specific antibodies ( red ) DAPI at 26 hpf . insets indicate representative from each immunostaining experiment red dots indicate EC aurka activation . Scale bars , 40 μm . of phospho - aurka - positive ECs ( H ) in equivalent fields of view for each MO - injected zebrafish in in ( G ) are result of three independent experiments with ≥ 10 per condition . top bottom whiskers represent maximum minimum values , respectively . * * P < 0 . 01 ( Unpaired Student ' s t - with Welch ' s correction ) .

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

Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 26 hpf. The insets indicate the representative area from each immunostaining experiment and the red dots indicate EC aurka activation. Scale bars, 40 μm. Quantification of the phospho-aurka-positive ECs (H) in equivalent fields of view for each MO-injected zebrafish in images in (G) are the result of three independent experiments with ≥ 10 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. **P < 0.01 (Unpaired Student's t-test with Welch's correction).


CEP41 - depleted hTERT - RPE1 were transfected with vectors encoding nothing ( MOCK ) , AURKA - T288D , or AURKA - K162R then immunostained with ARL13b - specific antibodies after serum starvation or serum retrieval . were taken from results of done 18 h after serum retrieval . Rectangles indicate representative that appear in magnified in right panels . Scale bars , 20 μm . A of ARL13b - labeled ciliated under indicated conditions in ( J ) . These are results of three independent experiments with ≥ 200 per condition ( mean ± SD ) . * P < 0 . 05 , * * * P < 0 . 01 , ns : non - significant ( Two - way ANOVA with Tukey ' s post hoc ) .

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

CEP41-depleted hTERT-RPE1 cells were transfected with vectors encoding nothing (MOCK), AURKA-T288D, or AURKA-K162R and then immunostained with ARL13b-specific antibodies after serum starvation or serum retrieval. Images were taken from the results of staining done 18 h after serum retrieval. Rectangles indicate the representative cells that appear in the magnified images in the right panels. Scale bars, 20 μm. A quantification of ARL13b-labeled ciliated cells under the indicated conditions in (J). These are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD). *P < 0.05, ***P < 0.01, ns: non-significant (Two-way ANOVA with Tukey's post hoc test).


Control CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , or AURKA - K162R subjected to an in vitro angiogenesis for 18 h under static ( K , L ) or shear stress ( M , N ) states . of tube node number in ( K , M ) tube in ( L , N ) from data examined within equivalent fields of view at each time point using ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition . Statistical significance was determined using two - way ANOVA followed by Tukey ' s post hoc ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : non - significant ) .

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

Control and CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, or AURKA-K162R and subjected to an in vitro angiogenesis assay for 18 h under static (K, L) or shear stress (M, N) states. Quantification of tube node number in (K, M) and tube length in (L, N) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant).


control or CEP41 siRNA - transfected HUVECs were cultivated under hypoxia ( 1 % O2 ) their whole lysates were used for immunoblot assays of CEP41 , phospho - AURKA , AURKA . resulting data were also compared to those of normoxic . were normalized against β - ACTIN in same blots .

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The control or CEP41 siRNA-transfected HUVECs were cultivated under hypoxia (1% O2) and their whole cell lysates were used for immunoblot assays of CEP41, phospho-AURKA, and AURKA. The resulting data were also compared to those of normoxic cells. Protein levels were normalized against β-ACTIN in the same blots.


VEGFA ( B ) VEGFR2 ( C ) mRNA were measured by qRT - PCR using cDNA from normoxic hypoxic controls or CEP41 - depleted . expression of GAPDH was quantified for normalization of those qRT - PCR results .

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VEGFA (B) and VEGFR2 (C) mRNA levels were measured by qRT-PCR using cDNA from normoxic and hypoxic controls or CEP41-depleted cells. The expression of GAPDH was quantified for the normalization of those qRT-PCR results.


Tg ( kdrl : eGFP ) zebrafish were injected with control - or cep41 - MOs then incubated under hypoxia at 28 hpf ( a stage of high shear stress ) for 2 h . They were subjected to immunostaining with phospho - AURKA - specific antibodies ( red ) DAPI at 30 hpf . insets indicate representative areas from each immunostaining red dots indicate activated aurka within ECs . Scale bars , 40 μm . of phospho - aurka - positive ECs ( E ) in equivalent fields of view for each MO - injected zebrafish in ( D ) are result of three independent experiments with ≥ 20 per condition . top bottom whiskers represent maximum minimum values , respectively . * P < 0 . 05 ( Unpaired Student ' s t - ) .

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

Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and then incubated under hypoxia at 28 hpf (a stage of high shear stress) for 2 h. They were subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 30 hpf. The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs. Scale bars, 40 μm. Quantification of phospho-aurka-positive ECs (E) in equivalent fields of view for each MO-injected zebrafish in (D) are the result of three independent experiments with ≥ 20 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. *P < 0.05 (Unpaired Student's t-test).


Zebrafish vegfa mRNA were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish subjected to either normoxia or hypoxia at 30 hpf . expression of zebrafish β - actin was quantified for normalization of these qRT - PCR results .

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Zebrafish vegfa mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.


vegfr2 ( G ) mRNA were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish subjected to either normoxia or hypoxia at 30 hpf . expression of zebrafish β - actin was quantified for normalization of these qRT - PCR results .

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vegfr2 (G) mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.


Control CEP41 - deficient HUVECs , transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , HIF1α , or HIF1α with CEP41 were cultivated under hypoxia subjected to an in vitro angiogenesis for 18 h . Scale bars , 400 μm .

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

Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm.


Control CEP41 - deficient HUVECs , transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , HIF1α , or HIF1α with CEP41 were cultivated under hypoxia subjected to an in vitro angiogenesis for 18 h . Scale bars , 400 μm . of tube node number in ( I ) tube in ( J ) from data examined within equivalent fields of view at each time point using ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm. Quantification of tube node number in (I) and tube length in (J) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia their whole lysates were used for immunoblot assays of HIF1α . HIF1α expression of hypoxic was compared to that of normoxic were normalized against β - ACTIN in same blot .

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HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia and their whole cell lysates were used for immunoblot assays of HIF1α. The HIF1α expression of hypoxic cells was compared to that of normoxic cells and the protein levels were normalized against β-ACTIN in the same blot.


Co - immunoprecipitation assays using HIF1α - , CEP41 - specific antibodies were performed in HEK 293T expressing GFP - CEP41 under normoxia or hypoxia . An IP using IgG - specific antibodies was performed as a negative control . NOR , normoxia ; HYP , hypoxia .

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Co-immunoprecipitation assays using HIF1α-, and CEP41-specific antibodies were performed in HEK 293T cells expressing GFP-CEP41 under normoxia or hypoxia. An IP using IgG-specific antibodies was performed as a negative control. NOR, normoxia; HYP, hypoxia.