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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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.


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

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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.


CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , HIF1α , or HIF1α with CEP41 exposed to hypoxia . VEGFA ( E ) VEGFR2 ( F ) mRNA were analyzed by qRT - PCR using cDNA from transfected . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined using one - way ANOVA followed by Tukey ' s post hoc ( * * * P < 0 . 001 , ns : non - significant ) . NOR , normoxia ; HYP , hypoxia .

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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 treated with MG132 or not , then were cultivated under hypoxia . Whole lysates were used for immunoblot assays for HIF1α , phospho - AURKA , AURKA , were normalized against β - ACTIN in same blots . NOR , normoxia ; HYP , hypoxia .

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

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.


of tube node number in ( H ) tube 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 .

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

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

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


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

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


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

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

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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 of relative expression of WOX5 in WT seu - 3 roots . Total 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 total number of scored samples . values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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 ) of niche areas in indicated genotypes at 5 DAG . Blue arrows indicate QC , red arrows indicate columella ( CSCs ) . numbers denote total number of scored samples , with similar showing in ( G - J ) . Scale bars : 20 μm . K of CSC layer in indicated genotypes at 5 DAG . Data Information ( K ) , data represent mean ± SD of three independent replicates . n denotes total number of scored samples . values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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

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 of QC184 β - glucuronidase ( GUS ) marker ( blue ) starch granules ( brown ) in WT ( L ) seu - 3 ( M ) seedlings at 5 DAG . In arrows indicate CSCs . Scale bars : 20 μm .

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 ) seu - 3 ( O ) seedlings at 5 DAG . P , Q Expression pattern of CS9227 in WT ( P ) seu - 3 ( Q ) seedlings at 5 DAG . In arrows indicate 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.


A - C Expression pattern of pWOX5 : : GFP in WT ( A ) , scr - 3 ( B ) plt1 - 4 plt2 - 2 ( C ) at 5 DAG . D of GFP in QC of pWOX5 : : GFP transgenic seedlings , as shown in ( A - C ) . GFP signal in each genotype was normalized relative to that in WT . Data information : In ( D ) n denotes total number of scored samples . 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 ( P < 0 . 01 ) .

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A-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 of relative expression of WOX5 in WT , scr - 3 , plt1 - 4 plt2 - 2 roots . Total was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data information : In ( E ) , * * P < 0 . 01 ( Student ' s t - ) . Scale bars : 20 µm .

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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 root of indicated genotypes at 5 DAG . Data information : In ( F ) n denotes total number of scored samples . 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 ( P < 0 . 01 ) .

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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 of indicated genotypes at 5 DAG . arrows indicate QC .

G-J Representative confocal images of the indicated genotypes at 5 DAG. White arrows indicate the QC.


K of GFP in QC of pSCR : : GFP pSCR : : GFP - SCR transgenic seedlings , as shown GFP signal of each genotype was normalized relative to that of WT . Data information ( K ) , n denotes total number of scored samples . values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . ( K ) , * * P < 0 . 01 ( Student ' s t - ) . Scale bars : 20 µm .

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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 niche of indicated genotypes at 5 DAG . dashed lines indicate QC region . numbers denote total number of scored samples , with similar showing in ( L - Q ) .

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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).


A Yeast two - hybrid ( Y2H ) assays showing interaction between SEU SCR . yeast transformants were plated on synthetic defined ( SD ) media lacking Leu Trp ( SD / - 2 ) or lacking Ade , His , Leu , Trp ( SD / - 4 ) to assess - interactions . AD , GAL4 activation domain ; BD , GAL4 - binding domain .

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A 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 . bound to amylose resin was eluted analyzed by immunoblotting using anti - FLAG antibody . asterisk indicates position of SEU - MBP .

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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 SEU in N . benthamiana leaves via Co - IP assays . SCR - GFP SEU - myc were transiently coexpressed in N . benthamiana leaves . samples were immunoprecipitated using anti - myc antibody .

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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 . extracts from WT SCR - GFP roots were isolated at 5 DAG immunoprecipitated with anti - GFP antibody .

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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 SEU physically bind to WOX5 promoter , as shown by ChIP - qPCR . was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , immunoprecipitated using anti - GFP antibody . precipitated was used as a template for qPCR . A , B , I indicated PCR amplicons ACT7 , control . Data information : In ( F ) , ( G ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - ) .

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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 SCR gene impairs recruitment of SEU to WOX5 promoter , as shown by ChIP - qPCR . was extracted from SEU - GFP SEU - GFP scr - 3 seedlings at 5 DAG precipitated with anti - GFP antibody . ChIP signals were quantified by qPCR as a of total input . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - ) .

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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 . pWOX5 : : LUC reporter was cotransformed with indicated effector constructs . pWOX5 : : LUC activity was normalized relative to internal control ( LUC / Renilla luciferase [ REN ] ) . schematic diagram shows construct used in transient expression assays . Arrows indicate promoter regions , boxes indicate coding sequences . Different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison ( P < 0 . 01 ) . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - ) .

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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 of indicated genotypes at 5 DAG . Scale bars : 20 μm . Insets show QC region in which solid lines indicate QC of WT , dashed lines indicate QC of seu - 3 . ( Insets scale bars : 5 μm ) . N of GFP signal in QC of WT seu - 3 seedlings expressing pWOX5 : : SEU - GFP pWOX5 : : SCR - GFP . GFP signal in seu - 3 seedlings was normalized relative to WT . values ( black dots ) are shown . n denotes total number of scored samples . Data information : In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - ) .

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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).


A Y2H assays showing that SEU interacts with SDG4 SDG25 . yeast transformants were plated on SD / - 2 SD / - 4 media to assess - interactions .

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A 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 WOX5 promoter , as shown by ChIP - qPCR . ACT7 , control . Data information was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . precipitated was used as a template for qPCR . In ( C ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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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 indicating that mutations in SDG4 SEU genes impair H3K4me3 deposition in WOX5 promoter . ChIP signal was normalized to WT . values ( black dots ) are shown . Data information : was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . precipitated was used as a template for qPCR . In ( D ) , ( E ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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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 indicated genotypes at 5 DAG . Scale bars : 20 µm . H of GFP in indicated genotypes expressing pWOX5 : : GFP . GFP signal in WT was set to 1 . n denotes 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 - ) .

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 of relative expression of WOX5 in WT ashr3 - 1 roots . Total was extracted from 5 mm root tip sections of seedlings at 5 DAG . 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 - ) .

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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 of niche in indicated genotypes at 5 DAG . Blue arrows indicate QC , red arrows indiate CSCs . numbers denote total number of scored samples , with same showing in ( J - M ) . Scale bars : 10 μm . N of CSC layer in 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 - ) .

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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).


A - G Representative confocal of indicated genotypes at 5 DAG . dashed lines indicate QC region . Scale bars : 20 μm .

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A-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 SDG4 . yeast transformants were plated on SD / - 2 SD / - 4 media to assess - interactions .

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 SDG4 in N . benthamiana leaves , as shown by Co - IP assays . SDG4 - myc , SCR - HA , SEU - GFP were coinfiltrated into N . benthamiana leaves . samples were immunoprecipitated with anti - GFP antibody immunoblotted with anti - myc anti - HA antibody . N . benthamiana leaves coinfiltrated with SDG4 - myc SCR - HA were used as a negative control .

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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 SDG4 in Arabidopsis . extracted from SDG4 - myc SDG4 - myc SCR - GFP were immunoprecipitated using anti - GFP antibody , immunoblotted using anti - myc anti - SEU antibodies .

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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.


D . expression showing top differentially expressed gene markers for each cluster of reference single atlas dataset . ( column ) are clustered by expression of main marker genes ( row ) .

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D. 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 genes Fas3 ( left panel ) zfh1 ( right panel ) used as markers to assign epithelial myoblast , respectively , in reference dataset .

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 orthogonal views of disc stained with anti - Zfh1 ( red ) anti - Fas3 ( green ) .

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 expression of marker genes identified for myoblast , epithelial tracheal across 26 clusters of reference atlas . Color represents average normalized expression level . Dot diameter represents fraction of expressing each gene in each cluster . Gene expression for each was normalized by total expression , then expression of each gene was scaled .

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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 orthogonal view of SPARC > GFP ( green ) stained with anti - Ct ( red ) 4 ´ , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Full genotype y - , w - / w - ; UAS - GFP / + ; SPARC - GAL4 / + .

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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/+.


D . Average expression level of genes used as known markers to assign each epithelial cluster in reference atlas dataset . colored by expression of nub , wg , zfh2 , Sox15 , grn , bnl , eyg , Ance , Idg4 drm .

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D. 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 expression of top marker genes identified for epithelial across 17 clusters of reference atlas dataset .

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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 ) counterstained with DAPI ( cyan ) . Scale bars : 100 μm . Full genotypes : P { CaryP } attP2 ( top bottom panels ) , P { PZ } dpp [ 10638 ] ; + , y - w - ; + ; P { lacW } Dad [ j1E4 ] .

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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].


B . Dot plots showing expression of top variable genes between IFM DFM myoblasts across 7 clusters of reference atlas dataset .

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B. Dot plots showing the expression levels of the top variable genes between IFM and DFM myoblasts across 7 clusters of the reference cell atlas dataset.


Confocal single plane of third instar larval wing discs stained with : ( C ) anti - Ct ( red ) anti - Zfh1 ( green ) , ( D ) kirre [ rp298 ] - lacZ ( anti - β - gal , green ) anti - Zfh1 ( red ) , ( E ) Ten - a [ MI04411 ] : : GFP ( green ) , anti - Ct ( red ) anti - Zfh1 ( cyan ) , ( F ) anti - Nrt ( red ) anti - Zfh1 ( cyan ) , ( G ) Argk [ CB3789 ] : : GFP ( green ) , anti - Ct ( red ) anti - Zfh1 ( cyan ) , ( H ) nkd [ MI00209 ] : : GFP ( green ) , anti - Ct ( red ) anti - Zfh1 ( cyan ) , ( I ) Ama [ NP1297 ] > GFP ( green ) , anti - Ct ( red ) anti - Zfh1 ( cyan ) ( J ) Mef2 > CD8 - GFP ( green ) , in situ hybridization of Ama - probe ( ) .

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Confocal single plane images of third instar larval wing discs stained with: (C) anti-Ct (red) and anti-Zfh1 (green), (D) kirre[rp298]-lacZ (anti-β-gal, green) and anti-Zfh1 (red), (E) Ten-a[MI04411]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (F) anti-Nrt (red) and anti-Zfh1 (cyan), (G) Argk[CB3789]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (H) nkd[MI00209]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (I) Ama[NP1297]>GFP (green), anti-Ct (red) and anti-Zfh1 (cyan) and (J) Mef2>CD8-GFP (green), and fluorescent in situ hybridization of Ama-RNA probe (white).


Confocal single plane of third instar larval wing discs stained with : K . Ama [ NP1297 ] > gTRACE showing lineage of Ama - GAL4 ( green ) active GAL4 ( red ) , in situ hybridization of Ama - probe ( ) . L . Confocal single plane of adult DFM of Ama [ NP1297 ] > gTRACE showing lineage of Ama - GAL4 ( green ) active GAL4 ( red ) stained with DAPI ( ) dorsal right , anterior up . DFM are numbered in following ( Lawrence , 1982 ) .

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Confocal single plane images of third instar larval wing discs stained with: K. Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red), and fluorescent in situ hybridization of Ama-RNA probe (white). L. Confocal single plane images of adult DFM of Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red) stained with DAPI (white) dorsal right, anterior up. DFM are numbered in white following (Lawrence, 1982).


A . Dot plot showing expression of top variable genes between myoblasts across 7 clusters of reference atlas dataset .

A. Dot plot showing the expression levels of the top variable genes between myoblasts across 7 clusters of the reference cell atlas dataset.


Confocal single plane of third instar larval wing discs stained with anti - Ct ( red ) ( B ) E ( spl ) m6 - BFM - gapGFP ( green ) , ( C ) E ( spl ) m7 - HLH [ VK00037 ] - GFP ( green )

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Confocal single plane images of third instar larval wing discs stained with anti-Ct (red) and (B) E(spl)m6-BFM-gapGFP (green), (C) E(spl)m7-HLH[VK00037]-GFP (green)


Confocal single plane of third instar larval wing discs stained with ( D ) E ( spl ) m3 - HLH [ GMR10E12 ] > GFP ( green ) .

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Confocal single plane images of third instar larval wing discs stained with (D) E(spl)m3-HLH[GMR10E12]>GFP (green).


H - I . Confocal single plane of third instar larval wing discs stained with anti - Ct ( cyan ) showing lineage of GAL4 ( green ) active GAL4 ( red ) in ( H ) E ( spl ) m3 - HLH [ GMR10E12 ] > gTRACE ( I ) E ( spl ) m6 - BFM - GAL4 > gTRACE .

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H-I. Confocal single plane images of third instar larval wing discs stained with anti-Ct (cyan) showing the lineage of GAL4 (green) and the active GAL4 (red) in (H) E(spl)m3-HLH[GMR10E12]>gTRACE and (I) E(spl)m6-BFM-GAL4>gTRACE.


A - E Confocal single plane of third instar larval wing discs stained with ( A ) anti - LamC ( green ) anti - Zfh1 ( red ) , ( B ) edl [ k12907 ] - lacZ ( green ) anti - Zfh1 ( red ) , ( C ) anti - H ( green ) anti - Zfh1 ( red ) , ( D ) Cg25C [ k00405 ] - lacZ ( green ) anti - Zfh1 ( red ) , or ( E ) vgk [ G00454 ] - GFP ( green ) anti - Ct ( red ) .

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A-E Confocal single plane images of third instar larval wing discs stained with (A) anti-LamC (green) and anti-Zfh1 (red), (B) edl[k12907]-lacZ (green) and anti-Zfh1 (red), (C) anti-H (green) and anti-Zfh1 (red), (D) Cg25C[k00405]-lacZ (green) and anti-Zfh1 (red), or (E) vgk[G00454]-GFP (green) and anti-Ct (red).


muscle - specific ( A - B ) Mef2 - GAL4 drivers were crossed to UAS - RNAi lines to knockdown expression of gene markers for myoblast clusters . ( A , Viability quantified by scoring of viable at each developmental stage , including early pupa ( black ) , pharate ( grey ) adult ( ) , relative to total number of . Data are expressed as stacked bars showing median with interquartile range ( n ≥ 3 independent biological replicates ) , unless noted as viable ( NQ ) lethal ( NQ ) , which stands for that were viable lethal , respectively , but no quantitative were performed . ( B , Flight ability scored by quantifying of flies landing on each section of column ( top , middle bottom ) as indicated on diagram on right . Data are expressed as stacked bars . total number of flies used in each was pooled together displayed next to each bar ( N ≥ 6 ) . n / a stands for non - applicable since are not viable .

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The muscle-specific (A-B) Mef2-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. (A, Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. (B, Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable.


muscle - specific 151 - GAL4 drivers were crossed to UAS - RNAi lines to knockdown expression of gene markers for myoblast clusters . C ) . Viability quantified by scoring of viable at each developmental stage , including early pupa ( black ) , pharate ( grey ) adult ( ) , relative to total number of . Data are expressed as stacked bars showing median with interquartile range ( n ≥ 3 independent biological replicates ) , unless noted as viable ( NQ ) lethal ( NQ ) , which stands for that were viable lethal , respectively , but no quantitative were performed . D ) . Flight ability scored by quantifying of flies landing on each section of column ( top , middle bottom ) as indicated on diagram on right . Data are expressed as stacked bars . total number of flies used in each was pooled together displayed next to each bar ( N ≥ 6 ) . n / a stands for non - applicable since are not viable .

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The muscle-specific 151-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. C). Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. D). Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable.


E . Confocal single plane of third instar larval wing discs from Mef2 > mCherry - RNAi , ( left panel ) Mef2 > chinmo [ HM04048 ] - RNAi ( right panel ) stained with anti - Zfh1 ( red ) DAPI ( cyan ) . Scale bars : 50 μm . F . of phenotype displayed in ( E ) . Stacked bars showing of wing discs scored as either normal or as abnormal . Data are expressed as mean ± , N = 32 discs per genotype , n = 3 independent experiments .

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E. Confocal single plane images of third instar larval wing discs from Mef2>mCherry-RNAi, (left panel) and Mef2>chinmo[HM04048]-RNAi (right panel) stained with anti-Zfh1 (red) and DAPI (cyan). Scale bars: 50 μm. F. Quantification of phenotype displayed in (E). Stacked bars showing the percentage of wing discs scored as either normal or as abnormal. Data are expressed as mean ± sem, N = 32 discs per genotype, n = 3 independent experiments.


G . of viability . Since UAS - Ama - RNAi [ GD12733 ] transgene is inserted on Chromosome X genotype of is Mef2 > Ama - RNAi whereas are Mef2 > + . Box plot showing of flies with genotype of interest ( ) over control flies ( ) . whiskers show 5 - 95 percentile , middle median . * p = 0 . 014 ( Mann - Whitney , N ≥ 6 independent biological replicates ) . Full genotypes are w , UAS - Dicer2 ; + ; mCherry - RNAi / Mef2 - GAL4 w , UAS - Dicer2 , UAS - Ama - RNAi [ GD12733 ] ; + ; Mef2 - GAL4 .

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G. Quantification of viability test. Since the UAS-Ama-RNAi[GD12733] transgene is inserted on Chromosome X the genotype of females is Mef2>Ama-RNAi whereas males are Mef2>+. Box plot showing the ratio of flies with genotype of interest (females) over control flies (males). The whiskers show 5-95 percentile, and the middle line the median. * p=0.014 (Mann-Whitney test, N≥6 independent biological replicates). Full genotypes are w, UAS-Dicer2;+; mCherry-RNAi/Mef2-GAL4 and w, UAS-Dicer2, UAS-Ama-RNAi[GD12733];+; Mef2-GAL4.


A . Lethality of 1151 > Ama - RNAi [ HMS00297 ] at pharate stage was partially rescued by overexpressing transgene Ama - cDNA . Representative of adults pharate pupa are shown . Full genotypes : 1151 - GAL4 ; UAS - myrRFP ; + , 1151 - GAL4 ; UAS - myrRFP ; UAS - Ama [ HMS00297 ] - RNAi , 1151 - GAL4 ; UAS - AmaOE / CyO ; UAS - Ama [ HMS00297 ] - RNAi 1151 - GAL4 ; UAS - AmaOE ; + .

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A. Lethality of 1151>Ama-RNAi[HMS00297] animals at pharate stage was partially rescued by overexpressing the transgene Ama-cDNA. Representative images of adults and pharate pupa are shown. Full genotypes: 1151-GAL4;UAS-myrRFP;+ , 1151-GAL4;UAS-myrRFP;UAS-Ama[HMS00297]-RNAi , 1151-GAL4;UAS-AmaOE/CyO;UAS-Ama[HMS00297]-RNAi and 1151-GAL4;UAS-AmaOE;+.


C . Average expression level of genes Fas3 ( left panel ) zfh1 ( right panel ) in 1151 > Ama - RNAi dataset .

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C. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) in the 1151>Ama-RNAi dataset.


D . Confocal single plane of 1151 > mCherry - RNAi 1151 > Ama - RNAi wing discs stained with anti - Ct ( red ) anti - Zfh1 ( green ) .

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D. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Ct (red) and anti-Zfh1 (green).


E . Dot plots showing expression of top markers for myoblast clusters identified in reference atlas across cluster of myoblasts in 1151 > Ama - RNAi 7 myoblast clusters in control dataset .

E. Dot plots showing the expression levels of the top markers for myoblast clusters identified in the reference cell atlas across the cluster of myoblasts in 1151>Ama-RNAi and 7 myoblast clusters in control dataset.


F . myoblast in 1151 > Ama - RNAi dataset projected into reference single atlas by transferring type labels using Seurat . bar graph represents total number of in each cluster normalized to total number of epithelial per genotype .

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F. The myoblast cells in 1151>Ama-RNAi dataset projected into the reference single cell atlas by transferring cell type labels using Seurat. The bar graph represents the total number of cells in each cluster normalized to the total number of epithelial cells per genotype.


G - H . Confocal single plane of 1151 > mCherry - RNAi 1151 > Ama - RNAi wing discs stained with anti - Zfh1 ( green ) ( G ) anti - Nrt ( ) at wandering third instar larval stage ( 110 - 135 h AEL ) or ( H ) DAPI ( red ) at early third instar larval wing discs ( 72 - 110 h AEL ) .

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G-H. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Zfh1 (green) and (G) anti-Nrt (white) at wandering third instar larval stage (110 - 135 h AEL) or (H) DAPI (red) at early third instar larval wing discs (72 - 110 h AEL).


Confocal single plane of 1151 > Ama - RNAi 1151 > mCherry - RNAi . A . Forming DFM at 40 h APF stained with anti - Zfh1 ( green ) , anti - Futsch ( 22c10 , red ) anti - Kettin ( ) . arrows point to wing hinge , wings pointing left , anterior up . B . Forming IFM ( DLM ) at 16 h APF , stained with anti - Futsch ( 22c10 , red ) , anti - Zfh1 ( green ) DAPI ( cyan ) . Yellow - dashed box indicates magnified ( bottom panel ) . Anterior up .

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Confocal single plane images of 1151>Ama-RNAi animals and 1151>mCherry-RNAi. A. Forming DFM at 40 h APF stained with anti-Zfh1 (green), anti-Futsch (22c10, red) and anti-Kettin (white). White arrows point to the wing hinge, wings pointing left, anterior up. B. Forming IFM (DLM) at 16 h APF, stained with anti-Futsch (22c10, red), anti-Zfh1 (green) and DAPI (cyan). Yellow-dashed box indicates magnified area (bottom panel). Anterior up.


C . DFM at 96 h APF stained with Phalloidin ( red ) , anti - Kettin ( green ) DAPI ( blue ) . Anterior up , dorsal right . DFM are numbered in as in ( Lawrence , 1982 ) . D . IFM at 96 h APF , transverse section ( top ) stained with anti - PS - integrin ( green ) , Phalloidin ( red ) DAPI ( blue ) , dorsal up ; sagittal section ( bottom ) stained with anti - Kettin ( green ) , Phalloidin ( red ) DAPI ( blue ) , anterior up , dorsal right .

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C. DFM at 96 h APF stained with Phalloidin (red), anti-Kettin (green) and DAPI (blue). Anterior up, dorsal right. DFM are numbered in white as in (Lawrence, 1982). D. IFM at 96 h APF, transverse section (top) stained with anti-PS-integrin (green), Phalloidin (red) and DAPI (blue), dorsal up; sagittal section (bottom) stained with anti-Kettin (green), Phalloidin (red) and DAPI (blue), anterior up, dorsal right.


C . Confirmation of sensor localization to different compartments . Green , HyPer7 ; blue , DAPI

C. Confirmation of sensor localization to different cellular compartments. Green, HyPer7; blue, DAPI stain


D . - H . Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A ( a III inhibitor , red curve data points ) or ethanol as control ( black data points ) . HEK293 were grown either with glucose or galactose as carbon source as indicated . Solid represents mean , points colored in lighter version of respective color are corresponding ratios measured in . Data information : As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * * , p ≤ 0 . 01 , * * * , p ≤ 0 .

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D.-H. Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A (a complex III inhibitor, red curve and data points) or ethanol as control (black line and data points). HEK293 cells were grown either with glucose or galactose as carbon source as indicated. Solid line represents mean, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. **, p ≤ 0.01, ***, p ≤ 0.001