figf2 ( 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 ) . ( 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 . ( 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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figf2(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).(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.(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.


figf3 ( 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 . ( 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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figf3(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.(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.


figf4 ( 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 ) . ( 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 . ( e ) Competitive binding assays using immobilized GST - Fbxo7 ( 1 - 398 ) incubated with IVT Flag - Parkin and / or full length ( top panel ) or N - terminally truncated ( bottom panel ) PINK1 - Myc . Input and bead - bound proteins were analyzed by immunoblotting as indicated . ( f ) As in e , immobilized GST - ΔN - PINK1 ( top panel ) , GST - Parkin ( middle panel ) and immobilized GST alone ( bottom panel ) were incubated with combinations of IVT Flag - Parkin , T7 - ΔN - PINK1 and Fbxo7 - HA as indicated . Input and bead - bound proteins were analyzed by immunoblotting with the indicated antibodies . All western blots were performed a minimum of three times . Full - length blots are presented in Supplementary Figure 9 .

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figf4(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).(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.(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.


figf5 ( 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 . ( 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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figf5(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.(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.


figf6 ( 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 ) . ( 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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figf6(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).(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.


figf7 ( 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 . ( 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 . ( 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 .

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figf7(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.(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.(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.


Figure 1HUVECs 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 ) . 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 .

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Figure 1HUVECs 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).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.


Figure 2Blood 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Blood 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).


Figure 3Control 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Control 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).


Figure 4HUVECs 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4HUVECs 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.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). **


Figure 5Control 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 ) . 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 ) ) . * 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Control 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).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)). *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)).


Figure 6VEGFA ( 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 . 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 ) . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6VEGFA (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.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).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).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).


Figure 7The 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7The 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.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.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.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.


Figure 8HUVECs 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 . 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 . 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 . 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 .

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

Figure 8HUVECs 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.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.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.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.


Figure 1A 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 ) . 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 ) . G - J Modified pseudo - Schiff propidium iodide ( mPS - PI ) staining of stem cell niche areas in the indicated genotypes at 5 DAG . Blue arrows indicate the QC , and red arrows indicate the columella stem cells ( CSCs ) . The numbers denote total number of scored samples , with similar phenotypes showing in ( G - J ) . Scale bars : 20 μm . K Quantification of the CSC layer in the indicated genotypes at 5 DAG . Data Information ( K ) , 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 ) . L , M Double staining of the QC184 β - glucuronidase ( GUS ) marker ( light blue ) and starch granules ( dark brown ) in WT ( L ) and seu - 3 ( M ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm . N , O Expression pattern of J2341 in WT ( N ) and seu - 3 ( O ) seedlings at 5 DAG . P , Q Expression pattern of CS9227 in WT ( P ) and seu - 3 ( Q ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm .

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

Figure 1A 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).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).G-J Modified pseudo-Schiff propidium iodide (mPS-PI) staining of stem cell niche areas in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indicate the columella stem cells (CSCs). The numbers denote total number of scored samples, with similar phenotypes showing in (G-J). Scale bars: 20 μm. K Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data Information (K), 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).L, M Double staining of the QC184 β-glucuronidase (GUS) marker (light blue) and starch granules (dark brown) in WT (L) and seu-3 (M) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.N, O Expression pattern of J2341 in WT (N) and seu-3 (O) seedlings at 5 DAG. P, Q Expression pattern of CS9227 in WT (P) and seu-3 (Q) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.


Figure 2A - C Expression pattern of pWOX5 : : GFP in WT ( A ) , scr - 3 ( B ) and plt1 - 4 plt2 - 2 ( C ) at 5 DAG . D Quantification of GFP fluorescence in the QC of pWOX5 : : GFP transgenic seedlings , as shown in ( A - C ) . GFP signal intensity in each genotype was normalized relative to that in the WT . Data information : In ( D ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( D ) different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . E RT - qPCR analysis of the relative expression levels of WOX5 in WT , scr - 3 , and plt1 - 4 plt2 - 2 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data information : In ( E ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm . F Primary root length of the indicated genotypes at 5 DAG . Data information : In ( F ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( F ) , different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . G - J Representative confocal images of the indicated genotypes at 5 DAG . White arrows indicate the QC . K Quantification of GFP fluorescence in the QC of pSCR : : GFP and pSCR : : GFP - SCR transgenic seedlings , as shown GFP signal intensity of each genotype was normalized relative to that of the WT . Data information ( K ) , n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . ( K ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm . L - Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG . White dashed lines indicate the QC region . The numbers denote total number of scored samples , with similar phenotypes showing in ( L - Q ) .

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

Figure 2A-C Expression pattern of pWOX5::GFP in WT (A), scr-3 (B) and plt1-4 plt2-2 (C) at 5 DAG. D Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic seedlings, as shown in (A-C). GFP signal intensity in each genotype was normalized relative to that in the WT. Data information: In (D) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (D) different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).E RT-qPCR analysis of the relative expression levels of WOX5 in WT, scr-3, and plt1-4 plt2-2 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data information: In (E) , **P < 0.01 (Student's t-test). Scale bars: 20 µm.F Primary root length of the indicated genotypes at 5 DAG. Data information: In (F) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (F), different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).G-J Representative confocal images of the indicated genotypes at 5 DAG. White arrows indicate the QC.K Quantification of GFP fluorescence in the QC of pSCR::GFP and pSCR::GFP-SCR transgenic seedlings, as shown GFP signal intensity of each genotype was normalized relative to that of the WT. Data information (K), n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. (K), **P < 0.01 (Student's t-test). Scale bars: 20 µm.L-Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG. White dashed lines indicate the QC region. The numbers denote total number of scored samples, with similar phenotypes showing in (L-Q).


Figure 3A Yeast two - hybrid ( Y2H ) assays showing the interaction between SEU and SCR . The yeast transformants were plated on synthetic defined ( SD ) media lacking Leu and Trp ( SD / - 2 ) or lacking Ade , His , Leu , and Trp ( SD / - 4 ) to assess protein - protein interactions . AD , GAL4 activation domain ; BD , GAL4 DNA - binding domain . B In vitro pull - down assays showing that SEU directly interacts with SCR but not with SHR . SCR - FLAG was pulled down by SEU - MBP immobilized on amylose resin . Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti - FLAG antibody . The asterisk indicates the position of SEU - MBP . C Verification of in vivo interactions between SCR and SEU in N . benthamiana leaves via Co - IP assays . SCR - GFP and SEU - myc were transiently coexpressed in N . benthamiana leaves . Protein samples were immunoprecipitated using anti - myc antibody . D Co - IP assays of SEU with SCR in Arabidopsis . Protein extracts from WT and SCR - GFP roots were isolated at 5 DAG and immunoprecipitated with anti - GFP antibody . F , G SCR and SEU physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP antibody . The precipitated DNA was used as a template for qPCR analysis . A , B , and I indicated the PCR amplicons ACT7 , control . Data information : In ( F ) , ( G ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) . H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was extracted from SEU - GFP and SEU - GFP scr - 3 seedlings at 5 DAG and precipitated with anti - GFP antibody . ChIP signals were quantified by qPCR as a percentage of total input DNA . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) . I SEU stimulated SCR - mediated WOX5 promoter activation in transient expression assays in N . benthamiana leaves . The pWOX5 : : LUC reporter was cotransformed with the indicated effector constructs . The pWOX5 : : LUC activity was normalized relative to the internal control ( LUC / Renilla luciferase [ REN ] ) . The schematic diagram shows the construct used in the transient expression assays . Arrows indicate promoter regions , and boxes indicate coding sequences . Different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) . J - M Representative confocal images of the indicated genotypes at 5 DAG . Scale bars : 20 μm . Insets show the QC region in which the solid white lines indicate the QC of WT , and the dashed white lines indicate the QC of seu - 3 . ( Insets scale bars : 5 μm ) . N Quantification of GFP signal intensity in the QC of WT and seu - 3 seedlings expressing pWOX5 : : SEU - GFP and pWOX5 : : SCR - GFP . GFP signal intensity in seu - 3 seedlings was normalized relative to the WT . Individual values ( black dots ) are shown . n denotes the total number of scored samples . Data information : In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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

Figure 3A Yeast two-hybrid (Y2H) assays showing the interaction between SEU and SCR. The yeast transformants were plated on synthetic defined (SD) media lacking Leu and Trp (SD/-2) or lacking Ade, His, Leu, and Trp (SD/-4) to assess protein-protein interactions. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain.B In vitro pull-down assays showing that SEU directly interacts with SCR but not with SHR. SCR-FLAG was pulled down by SEU-MBP immobilized on amylose resin. Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti-FLAG antibody. The asterisk indicates the position of SEU-MBP.C Verification of in vivo interactions between SCR and SEU in N. benthamiana leaves via Co-IP assays. SCR-GFP and SEU-myc were transiently coexpressed in N. benthamiana leaves. Protein samples were immunoprecipitated using anti-myc antibody.D Co-IP assays of SEU with SCR in Arabidopsis. Protein extracts from WT and SCR-GFP roots were isolated at 5 DAG and immunoprecipitated with anti-GFP antibody.F, G SCR and SEU physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP antibody. The precipitated DNA was used as a template for qPCR analysis. A, B, and I indicated the PCR amplicons ACT7, control. Data information: In (F), (G) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was extracted from SEU-GFP and SEU-GFP scr-3 seedlings at 5 DAG and precipitated with anti-GFP antibody. ChIP signals were quantified by qPCR as a percentage of total input DNA. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).I SEU stimulated SCR-mediated WOX5 promoter activation in transient expression assays in N. benthamiana leaves. The pWOX5::LUC reporter was cotransformed with the indicated effector constructs. The pWOX5::LUC activity was normalized relative to the internal control (LUC/Renilla luciferase [REN]). The schematic diagram shows the construct used in the transient expression assays. Arrows indicate promoter regions, and boxes indicate coding sequences. Different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).J-M Representative confocal images of the indicated genotypes at 5 DAG. Scale bars: 20 μm. Insets show the QC region in which the solid white lines indicate the QC of WT, and the dashed white lines indicate the QC of seu-3. (Insets scale bars: 5 μm). N Quantification of GFP signal intensity in the QC of WT and seu-3 seedlings expressing pWOX5::SEU-GFP and pWOX5::SCR-GFP. GFP signal intensity in seu-3 seedlings was normalized relative to the WT. Individual values (black dots) are shown. n denotes the total number of scored samples. Data information: In (N), data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


Figure 4A Y2H assays showing that SEU interacts with SDG4 and SDG25 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions . C SDG4 physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . ACT7 , control . Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( C ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . D , E ChIP - qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter . ChIP signal was normalized to the WT . Individual values ( black dots ) are shown . Data information : Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( D ) , ( E ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . F , G Expression pattern of pWOX5 : : GFP in the indicated genotypes at 5 DAG . Scale bars : 20 µm . H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5 : : GFP . GFP signal intensity in the WT was set to 1 . n denotes the total number of scored samples . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . I RT - qPCR analysis of the relative expression levels of WOX5 in WT and ashr3 - 1 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Individual values ( black dots ) are shown . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . J - M mPS - PI staining of stem cell niche in the indicated genotypes at 5 DAG . Blue arrows indicate the QC , and red arrows indiate the CSCs . The numbers denote total number of scored samples , with same phenotypes showing in ( J - M ) . Scale bars : 10 μm . N Quantification of the CSC layer in the indicated genotypes at 5 DAG . Data information In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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

Figure 4A Y2H assays showing that SEU interacts with SDG4 and SDG25. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.C SDG4 physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. ACT7, control. Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (C) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).D, E ChIP-qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter. ChIP signal was normalized to the WT. Individual values (black dots) are shown. Data information: Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (D), (E) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).F, G Expression pattern of pWOX5::GFP in the indicated genotypes at 5 DAG. Scale bars: 20 µm. H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5::GFP. GFP signal intensity in the WT was set to 1. n denotes the total number of scored samples. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).I RT-qPCR analysis of the relative expression levels of WOX5 in WT and ashr3-1 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Individual values (black dots) are shown. Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).J-M mPS-PI staining of stem cell niche in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indiate the CSCs. The numbers denote total number of scored samples, with same phenotypes showing in (J-M). Scale bars: 10 μm. N Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data information In (N), data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


Figure 5A - G Representative confocal images of the indicated genotypes at 5 DAG . The white dashed lines indicate the QC region . Scale bars : 20 μm . H Domain mapping of SEU involved in interactions with SCR and SDG4 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions . I SEU associates with SCR and SDG4 in N . benthamiana leaves , as shown by Co - IP assays . SDG4 - myc , SCR - HA , and SEU - GFP were coinfiltrated into N . benthamiana leaves . Protein samples were immunoprecipitated with anti - GFP antibody and immunoblotted with anti - myc and anti - HA antibody . N . benthamiana leaves coinfiltrated with SDG4 - myc and SCR - HA were used as a negative control . J Co - IP assays of SCR with SEU and SDG4 in Arabidopsis . Proteins extracted from SDG4 - myc and SDG4 - myc SCR - GFP plants were immunoprecipitated using anti - GFP antibody , and immunoblotted using anti - myc and anti - SEU antibodies .

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

Figure 5A-G Representative confocal images of the indicated genotypes at 5 DAG. The white dashed lines indicate the QC region. Scale bars: 20 μm.H Domain mapping of SEU involved in interactions with SCR and SDG4. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.I SEU associates with SCR and SDG4 in N. benthamiana leaves, as shown by Co-IP assays. SDG4-myc, SCR-HA, and SEU-GFP were coinfiltrated into N. benthamiana leaves. Protein samples were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-myc and anti-HA antibody. N. benthamiana leaves coinfiltrated with SDG4-myc and SCR-HA were used as a negative control.J Co-IP assays of SCR with SEU and SDG4 in Arabidopsis. Proteins extracted from SDG4-myc and SDG4-myc SCR-GFP plants were immunoprecipitated using anti-GFP antibody, and immunoblotted using anti-myc and anti-SEU antibodies.


Figure 1D . RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset . Cells ( column ) are clustered by the expression of the main marker genes ( row ) . E . Average expression level of the genes Fas3 ( left panel ) and zfh1 ( right panel ) used as markers to assign epithelial and myoblast cells , respectively , in the reference dataset . F . Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti - Zfh1 ( red ) and anti - Fas3 ( green ) . G . Dot plot showing the expression levels of the marker genes identified for the myoblast , epithelial and tracheal cells across the 26 clusters of the reference cell atlas . Color intensity represents the average normalized expression level . Dot diameter represents the fraction of cells expressing each gene in each cluster . Gene expression for each cell was normalized by the total expression , and then the expression of each gene was scaled . H . Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC > GFP ( green ) stained with anti - Ct ( red ) and 4 ´ , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Full genotype y - , w - / w - ; UAS - GFP / + ; SPARC - GAL4 / + .

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

Figure 1D. RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset. Cells (column) are clustered by the expression of the main marker genes (row).E. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) used as markers to assign epithelial and myoblast cells, respectively, in the reference dataset.F. Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti-Zfh1 (red) and anti-Fas3 (green).G. Dot plot showing the expression levels of the marker genes identified for the myoblast, epithelial and tracheal cells across the 26 clusters of the reference cell atlas. Color intensity represents the average normalized expression level. Dot diameter represents the fraction of cells expressing each gene in each cluster. Gene expression for each cell was normalized by the total expression, and then the expression of each gene was scaled.H. Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC>GFP (green) stained with anti-Ct (red) and 4´,6-diamidino-2-phenylindole (DAPI, blue). Full genotype y-, w-/w-; UAS-GFP/+; SPARC-GAL4/+.


Figure 2D . Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset . Cells colored by the expression of nub , wg , zfh2 , Sox15 , grn , bnl , eyg , Ance , Idg4 and drm . E . Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset . F . Confocal single plane image of third instar larval wing disc stained with anti - Nub , anti - Wg , anti - Svp , anti - Hairy ( red ) , reporting expression of dpp - lacZ , Dad - lacZ ( anti - β - gal , red ) and counterstained with DAPI ( cyan ) . Scale bars : 100 μm . Full genotypes : P { CaryP } attP2 ( top and bottom panels ) , P { PZ } dpp [ 10638 ] ; + , and y - w - ; + ; P { lacW } Dad [ j1E4 ] .

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

Figure 2D. Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset. Cells colored by the expression of nub, wg, zfh2, Sox15, grn, bnl, eyg, Ance, Idg4 and drm.E. Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset.F. Confocal single plane image of third instar larval wing disc stained with anti-Nub, anti-Wg, anti-Svp, anti-Hairy (red), reporting expression of dpp-lacZ, Dad-lacZ (anti-β-gal, red) and counterstained with DAPI (cyan). Scale bars: 100 μm. Full genotypes: P{CaryP}attP2 (top and bottom panels), P{PZ}dpp[10638];+, and y- w-;+; P{lacW}Dad[j1E4].