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

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


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

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


( e ) USP33 depletion leads to increased levels of p62 . HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time . Immunoblotting was performed using the indicated antibodies .

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(e) USP33 depletion leads to increased levels of p62. HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using the indicated antibodies.


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

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


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

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


( B ) Representative metaphase spreads from wild - type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control . Telomeric signals were detected with Cy3 - OO - ( CCCTAA ) 3 and are false colored in red , DNA is stained with DAPI and is false colored in cyan . Scale bar : 5µm .

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(B) Representative metaphase spreads from wild-type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control. Telomeric signals were detected with Cy3-OO-(CCCTAA)3 and are false colored in red, DNA is stained with DAPI and is false colored in cyan. Scale bar: 5µm.


( C ) Quantification of telomere fusions from experiment Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 3 , 000 telomeres counted / condition / experiment ) . ( * ) P < 0 . 05 ; unpaired two - tailed Student ' s t - test .

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(C) Quantification of telomere fusions from experiment Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>3,000 telomeres counted/condition/experiment). (*) P < 0.05; unpaired two-tailed Student's t-test.


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

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


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

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


( B ) Quantification of telomere fusions in SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days ( d7 , d8 , d11 ) . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 1 , 900 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 , ( * * ) P < 0 . 01 ; ( * ) P < 0 . 05 ; unpaired two - tailed Student ' s t - test .

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(B) Quantification of telomere fusions in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11). Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>1,900 telomeres counted/condition/experiment). (***) P < 0.001, (**) P< 0.01; (*) P< 0.05; unpaired two-tailed Student's t-test.


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

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


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

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


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

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


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

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B Representative images for CO-FISH on metaphase spreads in wild type (WT) Hela and U2OS cells. The leading strand telomeres were labeled by PNA-FISH with a (TTAGGG)3-Alexa488 probe (green), the lagging strand with a (CCCTAA)3-Cy5 probe (pink), and the DNA was stained with DAPI. Examples of telomere sister chromatid exchange (t-SCEs) events are marked with white arrows. Scale bar: 5µm


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

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


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

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(A-C) Representative images for detection of ATM pS9181, γH2AX, and 53BP1 at telomeres in wild-type (WT) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector (EV) control. Immunofluoresence (IF) for ATM pS1981 (gray), γH2AX (green) and 53BP1 (yellow) was combined with telomeric (CCCTAA)3-FISH (red) and DAPI staining total DNA. Scale bars: 5µm


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

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

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(E) Quantification of the number of 53BP1 TIFs per cell detected from three independent experiments. Data represent individual values per cell with the bar representing mean ± SD. (***) P < 0.001; unpaired two-tailed Student's t-test.


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

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


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

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(B) Representative images for Immunofluoresence (IF) detection of ATMpS1981 in wild-type HeLa cells γ-irradiated with a dose of 3Gy and harvested after the indicated times. Scale bar: 5µm


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

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


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

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


( B ) Representative metaphase spreads ( Scale bar : 5µm ) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 2 , 500 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 ; ( * * ) P < 0 . 01 unpaired two - tailed Student ' s t - test .

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(B) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,500 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01 unpaired two-tailed Student's t-test.


( C ) Representative metaphase spreads ( Scale bar : 5µm ) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi ( VE - 821 ) and quantification of telomere fusions . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 2 , 000 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 ; ( * * ) P < 0 . 01 ; ( * ) P < 0 . 05 unpaired two - tailed Student ' s t - test .

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(C) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi (VE-821) and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,000 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01; (*) P < 0.05 unpaired two-tailed Student's t-test.


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

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


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

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


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

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(E) Representative FACS plots of cells isolated from control (top) and kiaa1429 (RNAi) animals (bottom) show an increase in cell abundance in the X2 (Fincher et al, 2018) gate (green; Materials and Methods). (F) Quantification of the cells in each FACS gate in control and kiaa1429 (RNAi) animals showed a six-fold increase in the abundance of cells in the X2 gate (Student's t-test *** - p < 0.005. FACS experiments were performed in biological triplicates. Boxes represent the IQR, whiskers represent the 1.5 x IQR, and central band represents the med


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

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


( c ) Coimmunoprecipitation of endogenous Fbxo7 with Flag - Parkin in HEK293T cells transfected with Flag - Parkin or a control protein ( EGFP ) .

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(c) Coimmunoprecipitation of endogenous Fbxo7 with Flag-Parkin in HEK293T cells transfected with Flag-Parkin or a control protein (EGFP).


( d ) Flag immunoprecipitation was repeated in U2OS cells transfected with Flag - Parkin and either full - length ( 1 - 522 ) T7 - Fbxo7 or an N - terminal truncation lacking the Ubl domain ( 89 - 522 ) .

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(d) Flag immunoprecipitation was repeated in U2OS cells transfected with Flag-Parkin and either full-length (1-522) T7-Fbxo7 or an N-terminal truncation lacking the Ubl domain (89-522).


( e ) As in d , using lysates from U2OS cells expressing Flag - Parkin and T7 - Fbxo7 , either full length ( 1 - 522 ) or a C - terminal deletion of the proline - rich region ( 1 - 398 ) . * indicates IgG heavy chain .

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(e) As in d, using lysates from U2OS cells expressing Flag-Parkin and T7-Fbxo7, either full length (1-522) or a C-terminal deletion of the proline-rich region (1-398). * indicates IgG heavy chain.


( f ) In vitro - translated Flag - Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain ( 1 - 88 ) immobilized on glutathione beads . Bead - bound proteins and inputs were analyzed by immunoblotting with anti - Parkin antibodies .

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(f) In vitro-translated Flag-Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain (1-88) immobilized on glutathione beads. Bead-bound proteins and inputs were analyzed by immunoblotting with anti-Parkin antibodies.


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

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


( h ) Coimmunoprecipitation of Flag - Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins . CxVα and GAPDH are markers for mitochondria and cytoplasm , respectively . All western blots are representative of experiments performed at least three times . Full - length blots are presented in Supplementary Figure 9 .

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(h) Coimmunoprecipitation of Flag-Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins. CxVα and GAPDH are markers for mitochondria and cytoplasm, respectively. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.


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

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


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

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


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

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 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 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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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 O B-MEASURED_VAR 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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( 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 .

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(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 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 .

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

(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 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 .

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 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 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.


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 ) .

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

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 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

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 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 .

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 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

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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) . 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 ) .

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 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 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 ) .

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-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 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 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 ) .

O 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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 .

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 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 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.


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 .

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-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

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 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 ) . * *

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

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 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 ) .

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

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

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

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 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 .

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

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 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 ) ) . *

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 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 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

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 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 ) ) .

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 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 B-MEASURED_VAR 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 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 ) 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 ) .

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

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

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

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 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 .

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

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.


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 ) .

O O O O O B-MEASURED_VAR 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 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 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 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 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 ) .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 O B-CONTROLLED_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 O O O O O O 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 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 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 ) .

O 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 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).


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 .

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-MEASURED_VAR 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

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 ) 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 .

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

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 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 ) .

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 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-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 O O O O O O O O O O O O O O O O O O O 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 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 .

O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

B-MEASURED_VAR 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

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 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 .

O 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

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 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 .

O 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 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 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 .

O O 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

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 - , 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 .

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

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.


The CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , or HIF1α and cultivated under hypoxia . Whole cell lysates were used for immunoblot assays for phospho - AURKA and AURKA , and the resulting data were compared to those of normoxic and hypoxic control cells . Protein levels were normalized against β - ACTIN in the same blots . NOR , normoxia ; HYP , hypoxia .

O B-CONTROLLED_VAR O 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 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

The CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, or HIF1α and cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for phospho-AURKA and AURKA, and the resulting data were compared to those of normoxic and hypoxic control cells. Protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


The CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , HIF1α , or HIF1α with CEP41 and exposed to hypoxia . VEGFA ( E ) and VEGFR2 ( F ) mRNA levels were analyzed by qRT - PCR using cDNA from transfected cells . 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 ) . NOR , normoxia ; HYP , hypoxia .

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

The CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, HIF1α, or HIF1α with CEP41 and exposed to hypoxia. VEGFA (E) and VEGFR2 (F) mRNA levels were analyzed by qRT-PCR using cDNA from transfected cells. 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). NOR, normoxia; HYP, hypoxia.


Control or CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) or HIF1α and treated with MG132 or not , and then the cells were cultivated under hypoxia . Whole cell lysates were used for immunoblot assays for HIF1α , phospho - AURKA , and AURKA , and the protein levels were normalized against β - ACTIN in the same blots . NOR , normoxia ; HYP , hypoxia .

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

Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


Quantification of tube node number in ( H ) and tube length in ( I ) from in vitro angiogenesis assays with HIF1α # 1 or # 2 siRNAs - transfected HUVECs overexpressing nothing ( MOCK ) or CEP41 under hypoxia . 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 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

Quantification of tube node number in (H) and tube length in (I) from in vitro angiogenesis assays with HIF1α #1 or #2 siRNAs-transfected HUVECs overexpressing nothing (MOCK) or CEP41 under hypoxia. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


VEGFA ( J ) and VEGFR2 ( K ) mRNA levels were analyzed by qRT - PCR using cDNA from hypoxic HIF1α - depleted HUVECs transfected with control or CEP41 expression vectors . The results were compared to those of control and normoxic HIF1α - depleted cells . Data are shown as mean ± SD of three independent experiments .

B-MEASURED_VAR 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 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

VEGFA (J) and VEGFR2 (K) mRNA levels were analyzed by qRT-PCR using cDNA from hypoxic HIF1α-depleted HUVECs transfected with control or CEP41 expression vectors. The results were compared to those of control and normoxic HIF1α-depleted cells. Data are shown as mean ± SD of three independent experiments.


A Expression patterns of pSEU : : SEU - GFP and pWOX5 : : GFP in the embryos of the indicated genotypes at dermatogen , early globular , and heart stages . White arrows indicate the QC precursor cell , and white dashed lines indicate embryos . Scale bars : 10 µm .

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

A Expression patterns of pSEU::SEU-GFP and pWOX5::GFP in the embryos of the indicated genotypes at dermatogen, early globular, and heart stages. White arrows indicate the QC precursor cell, and white dashed lines indicate embryos. Scale bars: 10 µm.


B Quantification of pWOX5 : : GFP GFP fluorescence in wild - type ( WT ) and seu - 3 mutant embryos . Fluorescence intensity at the early globular stage of WT embryos was set to 1 . Data Information : In ( B ) data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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

B Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1. Data Information: In (B) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


C , D Expression pattern of pWOX5 : : GFP in WT ( C ) and seu - 3 ( D ) embryos at 5 days after germination ( DAG ) . Scale bars : 20 µm . E Quantification of GFP fluorescence in the QC of pWOX5 : : GFP transgenic plants , as shown in ( C ) and ( D ) . Fluorescence intensity was normalized to the WT . Data Information : ( E ) , data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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

C, D Expression pattern of pWOX5::GFP in WT (C) and seu-3 (D) embryos at 5 days after germination (DAG). Scale bars: 20 µm. E Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic plants, as shown in (C) and (D). Fluorescence intensity was normalized to the WT. Data Information: (E), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


F RT - qPCR analysis of the relative expression levels of WOX5 in WT and seu - 3 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data Information : ( F ) data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-qPCR analysis of the relative expression levels of WOX5 in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data Information: (F) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).