( d ) STUB1 knockdown inhibits proliferation . MRC - 5 fibroblasts ( 30 PDLs ) were seeded on six - well plates on day 0 ( 4 × 104 per well in 6 - well plate ; nine wells for each siRNA ) . were counted on days 1 , 4 7 ( three wells each ; mean ± s . d . ) , while siRNA transfection was performed on days 1 4 . * P0 . 01 ( Student ' s t - ) .

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(d) STUB1 knockdown inhibits cell proliferation. MRC-5 fibroblasts (30 PDLs) were seeded on six-well plates on day 0 (4 × 104 cells per well in 6-well plate; nine wells for each siRNA). Cells were counted on days 1, 4 and 7 (three wells each; mean±s.d.), while siRNA transfection was performed on days 1 and 4. *P0.01 (Student's t-test).


( e ) STUB1 knockdown induces senescence . siRNA transfection was performed as in c repeated 4 days later , followed by incubation for 3 days SA - β - Gal . Percentages of SA - β - Gal - positive were from triplicate experiments ( mean ± s . d . ) . * * P0 . 001 ( Student ' s t - ) .

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(e) STUB1 knockdown induces cellular senescence. The siRNA transfection was performed as in c and repeated 4 days later, followed by incubation for 3 days and SA-β-Gal staining. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).


( f , g ) STUB1 knockdown upregulates p21WAF1 IL - 8 . same set of as in e were examined in qRT - PCR for p21WAF1 mRNA ( f ) IL - 8 mRNA ( g ) . Data were from triplicate experiments ( mean ± s . d . ) . * * P0 . 001 ( Student ' s t - ) .

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(f,g) STUB1 knockdown upregulates p21WAF1 and IL-8. The same set of cells as in e were examined in qRT-PCR for p21WAF1 mRNA (f) and IL-8 mRNA (g). Data were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).


( h ) Δ133p53α rescues STUB1 knockdown - induced senescence . MRC - 5 fibroblasts were transfected with STUB1 siRNA ( no . 1 ) as in e . At next day after second transfection ( on day 5 ) , were transduced with a lentiviral vector driving Δ133p53α or its vector control . At 5 days after lentiviral transduction , were stained for SA - β - Gal activity . Representative pictures ( left ) quantitative data ( right ) are shown . Percentages of SA - β - Gal - positive were from triplicate experiments ( mean ± s . d . ) . * * P0 . 001 ( Student ' s t - ) .

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(h) Δ133p53α rescues STUB1 knockdown-induced senescence. MRC-5 fibroblasts were transfected with STUB1 siRNA (no. 1) as in e. At the next day after the second transfection (on day 5), the cells were transduced with a lentiviral vector driving Δ133p53α or its vector control. At 5 days after the lentiviral transduction, the cells were stained for SA-β-Gal activity. Representative pictures (left) and quantitative data (right) are shown. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P0.001 (Student's t-test).


( a ) STUB1 knockdown - induced repression of Δ133p53α is abrogated by bafilomycin A1 treatment . siRNA transfection in MRC - 5 fibroblasts was performed as in Fig . 3e . At 7 days after initial transfection , were untreated , treated with bafilomycin A1 ( 100 nM for 4 h ) or treated with MG - 132 ( 15 μM for 4 h ) , examined in immunoblots as indicated . results of Δ133p53α , full - p53 p62 / SQSTM1 in untreated replicate those shown in Fig . 3c . inhibition of autophagy by bafilomycin A1 was confirmed by increased of p62 / SQSTM1 LC3B . inhibition of proteasomal degradation by MG - 132 was confirmed by increased full - p53 .

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(a) STUB1 knockdown-induced repression of Δ133p53α is abrogated by bafilomycin A1 treatment. The siRNA transfection in MRC-5 fibroblasts was performed as in Fig. 3e. At 7 days after the initial transfection, the cells were untreated, treated with bafilomycin A1 (100 nM for 4 h) or treated with MG-132 (15 μM for 4 h), and examined in immunoblots as indicated. The results of Δ133p53α, full-length p53 and p62/SQSTM1 in untreated cells replicate those shown in Fig. 3c. The inhibition of autophagy by bafilomycin A1 was confirmed by increased levels of p62/SQSTM1 and LC3B. The inhibition of proteasomal degradation by MG-132 was confirmed by increased full-length p53.


( b ) STUB1 knockdown - induced repression of Δ133p53α is abrogated by knockdown of pro - autophagic . MRC - 5 fibroblasts ( PDLs 30 ) were transfected for 4 days with control siRNA ( − ) or STUB1 siRNA ( no . 1 , + ) , in combination with control siRNA ( c ) , p62 / SQSTM1 siRNA ( 62 ) , ATG5 siRNA ( A5 , no . 1 in Fig . 2a ) , ATG7 siRNA ( A7 , no . 1 in Fig . 2b ) or Beclin - 1 siRNA ( B1 , no . 2 in Fig . 2c ) , examined in immunoblots for indicated .

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(b) STUB1 knockdown-induced repression of Δ133p53α is abrogated by knockdown of pro-autophagic proteins. MRC-5 fibroblasts (PDLs 30) were transfected for 4 days with control siRNA (−) or STUB1 siRNA (no. 1, +), in combination with control siRNA (c), p62/SQSTM1 siRNA (62), ATG5 siRNA (A5, no. 1 in Fig. 2a), ATG7 siRNA (A7, no. 1 in Fig. 2b) or Beclin-1 siRNA (B1, no. 2 in Fig. 2c), and examined in immunoblots for indicated proteins.


( c ) Δ133p53α is recruited to autophagosomes upon STUB1 downregulation . MRC - 5 fibroblasts at early passage ( PDLs 30 ; Y ) , those at or close to replicative senescence ( PDLs 58 ; S ( Rep ) ) those induced to senesce by STUB1 knockdown ( S ( KD ) ) were used in immunoprecipitation ( IP ) with anti - STUB1 , anti - Hsp70 anti - LC3B antibodies , followed by immunoblots ( IB ) as indicated . Whole - lysates without IP ( input ) were also examined for Δ133p53α β - actin .

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(c) Δ133p53α is recruited to autophagosomes upon STUB1 downregulation. MRC-5 fibroblasts at early passage (PDLs 30; Y), those at or close to replicative senescence (PDLs 58; S (Rep)) and those induced to senesce by STUB1 knockdown (S (KD)) were used in immunoprecipitation (IP) with anti-STUB1, anti-Hsp70 and anti-LC3B antibodies, followed by immunoblots (IB) as indicated. Whole-protein lysates without IP (input) were also examined for Δ133p53α and β-actin levels.


( d ) Bafilomycin A1 stabilizes LC3B - interacting Δ133p53α in senescent . same set of as in c were treated with bafilomycin A1 ( + , 100 nM for 4 h ) or untreated ( − ) . These were used in IP with anti - LC3B antibody , followed by IB with anti - Δ133p53 antibody . Whole - lysates without IP were also examined in IB with anti - LC3B antibody . While untreated samples replicate result shown in c , differences between early passage senescent become more evident with bafilomycin A1 treatment .

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(d) Bafilomycin A1 stabilizes LC3B-interacting Δ133p53α in senescent cells. The same set of cells as in c were treated with bafilomycin A1 (+, 100 nM for 4 h) or untreated (−). These cells were used in IP with anti-LC3B antibody, followed by IB with anti-Δ133p53 antibody. Whole-protein lysates without IP were also examined in IB with anti-LC3B antibody. While untreated samples replicate the result shown in c, the differences between early passage and senescent cells become more evident with bafilomycin A1 treatment.


( a ) Δ133p53α is polyubiquitinated . FLAG - tagged versions of wild - type Δ133p53α ( wt ) mutant Δ133p53α ( mut ; from c below ) , along with vector control ( − ) , were retrovirally transduced into MRC - 5 fibroblasts . wild - type Δ133p53α - expressing had a pair of STUB1 siRNA - transfected ( + , siRNA no . 1 for 4 days ) untransfected ( − ) counterparts . These were maintained for 8 h under acid - serum - starved conditions with bafilomycin A1 ( 100 nM ) . lysates were used either in IP with anti - ubiquitin ( Ub ) antibody , followed by IB with anti - FLAG antibody ( top ) , or directly in IB for FLAG - Δ133p53α , STUB1 β - actin ( lower three panels ) . Smear signals indicate polyubiquitinated FLAG - Δ133p53α ( Poly - Ub ) . Asterisk indicates a nonspecific band in negative control . These experimental conditions resulted in similar amounts of FLAG - Δ133p53α in presence absence of STUB1 knockdown , allowing quantitative of ubiquitination . relative densitometric values of wild - type FLAG - Δ133p53α polyubiquitination in presence absence of STUB1 knockdown ( normalized with total FLAG - Δ133p53α ) are shown . (

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(a) Δ133p53α protein is polyubiquitinated. FLAG-tagged versions of wild-type Δ133p53α (wt) and mutant Δ133p53α (mut; from c below), along with vector control (−), were retrovirally transduced into MRC-5 fibroblasts. The wild-type Δ133p53α-expressing cells had a pair of STUB1 siRNA-transfected (+, siRNA no. 1 for 4 days) and untransfected (−) counterparts. These cells were maintained for 8 h under amino acid- and serum-starved conditions with bafilomycin A1 (100 nM). Protein lysates were used either in IP with anti-ubiquitin (Ub) antibody, followed by IB with anti-FLAG antibody (top), or directly in IB for FLAG-Δ133p53α, STUB1 and β-actin (lower three panels). Smear signals indicate polyubiquitinated FLAG-Δ133p53α protein (Poly-Ub). Asterisk indicates a nonspecific band in negative control. These experimental conditions resulted in similar amounts of FLAG-Δ133p53α in the presence and absence of STUB1 knockdown, allowing quantitative analysis of ubiquitination. The relative densitometric values of wild-type FLAG-Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown (normalized with total FLAG-Δ133p53α) are shown. (


( b ) Δ133p53α is ubiquitinated at C - terminal lysine residues . MRC - 5 fibroblasts expressing wild - type FLAG - Δ133p53α in absence of STUB1 knockdown were used in IP with anti - FLAG antibody , resulting immunoprecipitates were analysed using mass spectrometry . Representative MS / MS spectrum of an [ M + 3H ] 3 + peptide from FLAG - Δ133p53α shows sites of ubiquitin modification . peptide for spectrum is shown , with sites of modification marked as asterisks : - acid positions 248 249 , which correspond to positions 381 382 in full - p53 . same peptide was also observed in unmodified state .

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(b) Δ133p53α is ubiquitinated at the C-terminal lysine residues. MRC-5 fibroblasts expressing wild-type FLAG-Δ133p53α in the absence of STUB1 knockdown were used in IP with anti-FLAG antibody, and the resulting immunoprecipitates were analysed using mass spectrometry. Representative MS/MS spectrum of an [M+3H]3+ peptide from FLAG-Δ133p53α shows sites of ubiquitin modification. The peptide sequence for the spectrum is shown, with the sites of modification marked as asterisks: amino-acid positions 248 and 249, which correspond to positions 381 and 382 in full-length p53. The same peptide was also observed in the unmodified state.


( c ) Lysine ( K ) - to - arginine ( R ) substitutions at - acid residues 248 249 .

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(c) Lysine (K)-to-arginine (R) substitutions at amino-acid residues 248 and 249.


( d ) Mutation of ubiquitination sites renders Δ133p53α resistant to degradation . same three as in a were cultured under - acid - serum - starved conditions for 8 h ( without bafilomycin A1 ) , transfected with STUB1 siRNA ( no . 1 for 4 days ) or untreated , examined in IBs for FLAG - Δ133p53α , STUB1 , p62 / SQSTM1 β - actin . effect of starvation was confirmed by decreased p62 / SQSTM1 in each . upregulation of p62 / SQSTM1 by wild - type mutant FLAG - Δ133p53α is likely associated with enhanced proliferation by Δ133p53α overexpression .

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(d) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation. The same three cells as in a were cultured under amino-acid- and serum-starved conditions for 8 h (without bafilomycin A1), transfected with STUB1 siRNA (no. 1 for 4 days) or untreated, and examined in IBs for FLAG-Δ133p53α, STUB1, p62/SQSTM1 and β-actin. The effect of starvation was confirmed by decreased p62/SQSTM1 in each cell. The upregulation of p62/SQSTM1 by wild-type and mutant FLAG-Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression.


( a ) Δ133p53α is colocalized with p62 / SQSTM1 upon autophagy induction . MRC - 5 fibroblasts expressing FLAG - Δ133p53α ( wild - type ) were cultured under - acid - serum - starved conditions for 4 h , co - immunostained with anti - FLAG antibody ( Δ133p53α , green ) anti - p62 / SQSTM1 antibody ( red ) . Two representative sets of , including with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) merged , are shown . Scale bars , 10 μm .

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(a) Δ133p53α is colocalized with p62/SQSTM1 upon autophagy induction. MRC-5 fibroblasts expressing FLAG-Δ133p53α (wild-type) were cultured under amino-acid- and serum-starved conditions for 4 h, and co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-p62/SQSTM1 antibody (red). Two representative sets of images, including nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and merged images, are shown. Scale bars, 10 μm.


( b ) Knockdown of p62 / SQSTM1 stabilizes endogenous Δ133p53α . MRC - 5 fibroblasts at late passage ( PDLs 51 ) were transfected with p62 / SQSTM1 siRNA ( p62 ) or control siRNA ( C ) for 3 days , examined in immunoblots for Δ133p53α p62 / SQSTM1 expression . relative expression of Δ133p53α ( normalized with β - actin ) are shown .

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(b) Knockdown of p62/SQSTM1 stabilizes endogenous Δ133p53α. MRC-5 fibroblasts at late passage (PDLs 51) were transfected with p62/SQSTM1 siRNA (p62) or control siRNA (C) for 3 days, and examined in immunoblots for Δ133p53α and p62/SQSTM1 expression. The relative expression levels of Δ133p53α (normalized with β-actin) are shown.


( c ) Δ133p53α is colocalized with LC3B upon autophagy induction . MRC - 5 fibroblasts with FLAG - Δ133p53α under starved conditions ( as in a ) were co - immunostained with anti - FLAG antibody ( Δ133p53α , green ) anti - LC3B antibody ( red ) . Two representative sets of , including DAPI merged , are shown . Scale bars , 10 μm . ( d ) Quantitative of Δ133p53α - LC3B colocalization . number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 . Data ( colocalization ) are mean ± s . d . Almost Δ133p53α signals were colocalized with LC3B , while ~ 80 % of LC3B signals were colocalized with Δ133p53α .

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(c) Δ133p53α is colocalized with LC3B upon autophagy induction. MRC-5 fibroblasts with FLAG-Δ133p53α under starved conditions (as in a) were co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-LC3B antibody (red). Two representative sets of images, including DAPI and merged images, are shown. Scale bars, 10 μm. (d) Quantitative analysis of Δ133p53α-LC3B colocalization. The number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 cells. Data (percent colocalization) are mean±s.d. Almost all Δ133p53α signals were colocalized with LC3B, while ~80% of LC3B signals were colocalized with Δ133p53α.


( A ) β - Catenin is hyperphosphorylated during mitotic phase . HeLa CCL2 were synchronized at G1 / S phase using a double thymidine block released using medium containing 200 ng / mL nocodazole ( Noc . ) . were harvested at indicated time points treated with ( + ) or without ( - ) λ - phosphatase ( λ - PPase ) . resulting lysates were used for immunoblot . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE . Asterisk indicates phosphorylated form of Cdc25C .

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(A) β-Catenin is hyperphosphorylated during the mitotic phase. HeLa CCL2 cells were synchronized at the G1/S phase using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.). Cells were harvested at the indicated time points and treated with (+) or without (-) λ-phosphatase (λ-PPase). The resulting cell lysates were used for immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. Asterisk indicates the phosphorylated form of Cdc25C.


( F ) Total lysates from nocodazole ( Noc . ) - treated HeLa CCL2 were immunoprecipitated with two different Plk1 antibodies . Each antibody ( gifted from Kyung S . Lee , NIH / NCI ) recognized either Plk1 N - terminus ( N - term . Ab . ) or C - terminus ( C - term . Ab . ) . resulting precipitates were subjected to immunoblot . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE .

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(F) Total cell lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were immunoprecipitated with two different Plk1 antibodies. Each antibody (gifted from Kyung S. Lee, NIH/NCI) recognized either Plk1 N-terminus (N-term. Ab.) or C-terminus (C-term. Ab.). The resulting precipitates were subjected to immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.


( G ) Lysates from nocodazole ( Noc . ) - treated HeLa CCL2 were mixed with either control ( phosphate - buffered saline only , + PBS ) , 10 μg / ml of high - affinity Plk1 PBD binding phospho - peptide ( PLHSpT , + P ) , or 10 μg / ml of non - phospho - peptide ( PLHST , + NP ) . resulting lysates were immunoprecipitated with anti - Plk1 antibody . Precipitates were subjected to immunoblot . Immunoblotting with anti - Dvl2 antibody represents a positive control of phospho - peptide ( PLHSpT , + P ) competition for Plk1 binding . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE .

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(G) Lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were mixed with either control (phosphate-buffered saline only, +PBS), 10 μg/ml of high-affinity Plk1 PBD binding phospho-peptide (PLHSpT, +P), or 10 μg/ml of non-phospho-peptide (PLHST, +NP). The resulting cell lysates were immunoprecipitated with anti-Plk1 antibody. Precipitates were subjected to immunoblot analysis. Immunoblotting with anti-Dvl2 antibody represents a positive control of phospho-peptide (PLHSpT, +P) competition for Plk1 binding. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.


( C ) Bacterially produced β - catenin T1 WT , T3 WT , corresponding alanine ( " A " ) mutants were purified reacted with Flag - Plk1 WT in presence of [ γ - 32P ] ATP . Incorporation of 32P onto each " A " mutant of β - catenin was confirmed by SDS - PAGE followed by autoradiography .

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(C) Bacterially produced β-catenin T1 WT, T3 WT, and corresponding alanine ("A") mutants were purified and reacted with Flag-Plk1 WT in the presence of [γ-32P]ATP. Incorporation of 32P onto each "A" mutant of β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.


( H ) HeLa CCL2 were treated for 18 h with 200 ng / mL nocodazole followed by treatment with either DMSO ( control ) or BI 2536 for an additional 1 h . resulting were lysed with 2 × Laemmli SDS sample buffer used for immunoblot with indicated antibodies .

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(H) HeLa CCL2 cells were treated for 18 h with 200 ng/mL nocodazole followed by treatment with either DMSO (control) or BI 2536 for an additional 1 h. The resulting cells were lysed with 2× Laemmli SDS sample buffer and used for immunoblot analysis with the indicated antibodies.


( C ) Asynchronously growing HeLa CCL2 were immunostained with indicated antibodies as described in ( A ) , midbody localization of β - catenin / β - catenin p - S60 in late telophase was observed using a confocal microscope ( Zeiss LSM 700 ) . Scale bars , 10 μm .

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(C) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies as described in (A), and the midbody localization of β-catenin/β-catenin p-S60 in late telophase cells was observed using a confocal laser microscope (Zeiss LSM 700). Scale bars, 10 μm.


( A ) Asynchronously growing HeLa CCL2 were immunostained with indicated antibodies , early telophase were observed using a super - resolution confocal scanning microscope ( LSM - 880 with Airyscan ) ( left panel ) . Scale bars , 10 μm . Linear signal profiles of β - catenin / Plk1 / β - catenin p - S60 / F - actin in contractile ring region from XY plane of immunostained early telophase ( right panel ) . immunofluorescence linear signal profiles show representative data of more than 50 telophase obtained from three independent experiments . value of relative neck thickness is shown in lower left corner of merged ( A ) .

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(A) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies, and early telophase cells were observed using a super-resolution confocal laser scanning microscope (LSM-880 with Airyscan) (left panel). Scale bars, 10 μm. Linear signal profiles of β-catenin/Plk1/β-catenin p-S60/F-actin in the contractile ring region from the XY plane of immunostained early telophase cells (right panel). All immunofluorescence images and linear signal profiles show representative data of more than 50 telophase cells obtained from three independent experiments. The value of the relative neck thickness is shown in the lower left corner of the merged images (A).


HeLa CCL2 stably expressing control vector only ( + vector ) , β - catenin WT ( + WTR ) , S60A mutant ( + S60AR ) , or S60D mutant ( + S60DR ) were generated using lentiviral infection then infected with β - catenin knockdown lentivirus ( shβ - catenin ) . A dot density graph with a box plot shows ( % ) of with cytokinesis - defective ( C ) . box plot represents : central blue band , Median ; box , Interquartile range ; upper whisker , Maximum observation below upper fence ; lower whisker , Minimum observation above lower fence ; dot , Outlier . Each symbol in graph represents a of more than 30 from three independent experiments . * * * , p < 0 . 001 ( one - way ANOVA ) ; Scale bars , 10 μm .

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HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). A dot density graph with a box plot shows the percentage (%) of cells with cytokinesis-defective phenotypes (C). The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Each symbol in the graph represents a percentage of more than 30 cells from three independent experiments. ***, p<0.001 (one-way ANOVA); Scale bars, 10 μm.


HeLa CCL2 transiently transfected with either GFP - β - catenin WT or GFP - β - catenin S60A were monitored during cycle progression by live imaging ( E ) . subcellular localizations of GFP - tagged β - catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti - GFP antibody . were additionally stained for Plk1 ( see Appendix Figure S5A ) . specific localization of GFP - β - catenin WT at midbody was observed under a confocal microscope with high magnification ( E , left panel ) . Cytokinesis - defective in each transfected HeLa were monitored ( E , right panel )

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HeLa CCL2 cells transiently transfected with either GFP-β-catenin WT or GFP-β-catenin S60A were monitored during cell cycle progression by live cell imaging (E). The subcellular localizations of GFP-tagged β-catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti-GFP antibody. The cells were additionally stained for Plk1 (see Appendix Figure S5A). The specific localization of GFP-β-catenin WT at the midbody was observed under a confocal laser microscope with high magnification (E, left panel). Cytokinesis-defective cellular phenotypes in each transfected HeLa cell were monitored (E, right panel)


( E , F ) HeLa CCL2 infected with indicated lentiviruses were co - transfected with HA - Ect2 Flag - Plk1 . were then treated with nocodazole ( 200 ng / mL ) for 18 h released from cycle arrest by incubation in fresh medium for 6 h . resulting were used for immunoprecipitation using anti - Flag ( E ) or anti - HA ( F ) antibodies . Precipitated beads were used for immunoblot using indicated antibodies .

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(E, F) HeLa CCL2 cells infected with the indicated lentiviruses were co-transfected with HA-Ect2 and Flag-Plk1. Cells were then treated with nocodazole (200 ng/mL) for 18 h and released from cell cycle arrest by incubation in fresh medium for 6 h. The resulting cells were used for immunoprecipitation analysis using anti-Flag (E) or anti-HA (F) antibodies. Precipitated beads were used for immunoblot analysis using the indicated antibodies.


( G , H ) HeLa CCl2 infected with either sh - control luciferase ( shGL ) or shβ - catenin lentiviruses were co - immunostained with anti - β - catenin anti - Ect2 antibodies . Localization of β - catenin Ect2 in midbody of telophase was observed using a confocal microscope ( G ) signal of Ect2 was monitored in midbody of each knockdown telophase ( H ) . Arrows indicate total β - catenin Ect2 located at midbody in telophase HeLa CCL2 ( G ) . A dot density graph with a box plot shows signal of midbody - localized Ect2 in telophase . box plot represents : central blue band , Median ; box , Interquartile range ; upper whisker , Maximum observation below upper fence ; lower whisker , Minimum observation above lower fence ; dot , Outlier . Approximately 70 from three independent experiments were measured . * * * , p < 0 . 001 ( unpaired two - t ) ; Scale bars , 10 μm . value of relative neck thickness is shown in lower left corner of merged ( G ) .

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(G, H) HeLa CCl2 cells infected with either sh-control luciferase (shGL) or shβ-catenin lentiviruses were co-immunostained with anti-β-catenin and anti-Ect2 antibodies. Localization of β-catenin and Ect2 in the midbody of telophase cells was observed using a confocal microscope (G) and the signal intensity of Ect2 was monitored in the midbody of each knockdown telophase cell (H). Arrows indicate total β-catenin and Ect2 located at the midbody in telophase HeLa CCL2 cells (G). A dot density graph with a box plot shows the signal intensity of midbody-localized Ect2 in telophase cells. The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Approximately 70 cells from three independent experiments were measured. ***, p<0.001 (unpaired two-tailed t test); Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images (G).


HeLa CCL2 treated with DMSO ( control ) or Plk1 inhibitor , BI 2536 ( 1 μM ) for 20 min were immunostained with a combination of either anti - total β - catenin anti - Ect2 antibodies or anti - β - catenin p - S60 anti - Ect2 antibodies ( A ) . Scale bars , 10 μm .

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HeLa CCL2 cells treated with DMSO (control) or the Plk1 inhibitor, BI 2536 (1 μM) for 20 min were immunostained with a combination of either anti-total β-catenin and anti-Ect2 antibodies or anti-β-catenin p-S60 and anti-Ect2 antibodies (A). Scale bars, 10 μm.


( C ) After 18 h of nocodazole ( 200 ng / mL ) - induced cycle arrest , HeLa CCL2 transfected with HA - Ect2 were released by incubation in fresh medium for 2 h treated with either DMSO ( control ) or Plk1 inhibitor ( BI 2536 ) for indicated times . resulting were used for immunoprecipitation using anti - HA antibody , immunoprecipitates were separated by SDS - PAGE followed by immunoblot with indicated antibodies . band of β - catenin bound to HA - Ect2 was quantified with ImageJ normalized to signal of immunoprecipitated HA - Ect2 , relative values are shown below bands .

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(C) After 18 h of nocodazole (200 ng/mL)-induced cell cycle arrest, HeLa CCL2 cells transfected with HA-Ect2 were released by incubation in fresh medium for 2 h and treated with either DMSO (control) or Plk1 inhibitor (BI 2536) for the indicated times. The resulting cells were used for immunoprecipitation analysis using anti-HA antibody, and immunoprecipitates were separated by SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The band intensity of β-catenin bound to HA-Ect2 was quantified with ImageJ and normalized to the signal intensity of immunoprecipitated HA-Ect2, and the relative values are shown below the bands.


( G ) HeLa CCL2 were transfected with either wild type Flag − β - catenin ( + WT ) , Flag - β - catenin S60A mutant ( + S60A ) , or Flag - β - catenin S60D mutant ( + S60D ) , then treated with 200 ng / mL nocodazole for 18 h ( metaphase ) released by incubation in fresh medium for 2 h ( telophase ) . lysates were used for pull - down with rhotekin - RBD beads , pulled down beads were separated by 12 % SDS - PAGE followed by immunoblot with indicated antibodies . was then stained with Coomassie ( CBB ) to determine amount of RBD precipitated .

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(G) HeLa CCL2 cells were transfected with either wild type Flag−β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D), then treated with 200 ng/mL nocodazole for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were used for pull-down analysis with rhotekin-RBD beads, and the pulled down beads were separated by 12 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The membrane was then stained with Coomassie (CBB) to determine the amount of RBD precipitated.


Left : Representative time - lapse showing migration of single hRPE on surface of polyacrylamide gels with uniform stiffness ( top ) stiffness gradient ( bottom ) , referring as random migration durotaxis migration respectively . Yellow is outline blue is migration trajectory . Scale bar : 50 μm . Middle : Angular displacement of hRPE on uniform stiffness gels ( top ) stiffness gradient gels ( bottom ) . Right : y / x FMI ( forward migration index ) , velocity persistence of hRPE with random migration durotaxis .

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Left: Representative time-lapse images showing the migration of single hRPE cells on the surface of polyacrylamide gels with uniform stiffness (top) and stiffness gradient (bottom), referring as random migration and durotaxis migration respectively. Yellow line is the cell outline and blue line is the cell migration trajectory. Scale bar: 50 μm. Middle: Angular displacement of hRPE cells on the uniform stiffness gels (top) and stiffness gradient gels (bottom). Right: y/x FMI (forward migration index), velocity and persistence of hRPE cells with random migration and durotaxis.


Left : Representative immunofluorescent of F - actin in control CK - 666 ( 100 μM , 3 h ) treated hRPEs . Scale bar : 20 μm . Right : Y FMI of control CK - 666 ( 100 μM , pre - treated for 1h incubated for whole experiment ) treated hRPEs moving on stiffness gradient gel .

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Left: Representative immunofluorescent staining of F-actin in control and CK-666 (100 μM, 3 h) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and CK-666 (100 μM, pre-treated for 1h and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.


Left : Representative immunofluorescent of α - tubulin in control nocodazole ( 8 μM , 30 min ) treated hRPEs . Scale bar : 20 μm . Right : Y FMI of control nocodazole ( 8 μM , pre - treated for 10 min incubated for whole experiment ) treated hRPEs moving on stiffness gradient gel .

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Left: Representative immunofluorescent staining of α-tubulin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and nocodazole (8 μM, pre-treated for 10 min and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.


Representative immunofluorescent of γ - tubulin ( yellow ) DAPI ( blue ) in control centrinone - B ( 500 nM , 7 days ) treated hRPEs .

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Representative immunofluorescent staining of γ-tubulin (yellow) and DAPI (blue) in control and centrinone-B (500 nM, 7 days) treated hRPEs.


Angular displacement y FMI of control centrinone - B ( 500 nM , 7 days ) treated hRPEs moving on stiffness gradient gels .

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Angular displacement and y FMI of control and centrinone-B (500 nM, 7 days) treated hRPEs moving on stiffness gradient gels.


Western blot showing level of CAMSAP2 in negative control CAMSAP2 siRNA transfected hRPEs . GAPDH was used as loading control . Bar chart shows of normalized to GAPDH in each condition .

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Western blot showing the protein level of CAMSAP2 in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH was used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.


Angular displacement y FMI of negative control CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels .

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Angular displacement and y FMI of negative control and CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels.


Western blot showing level of AKAP450 in negative control AKAP450 siRNA transfected hRPEs . α - tubulin was used as loading control . Bar chart shows of normalized to α - tubulin in each condition .

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Western blot showing the protein level of AKAP450 in negative control and AKAP450 siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition.


Angular displacement y FMI of control AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels .

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Angular displacement and y FMI of control and AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels.


Left : Representative immunofluorescent of α - tubulin in control , CAMSAP2 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . Scale bar : 20 μm . Right : of microtubule .

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Left: Representative immunofluorescent staining of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of microtubule fluorescence intensity.


Western blot showing level of α - tubulin in control , CAMSAP2 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . GAPDH is used as loading control . Bar chart shows of normalized to GAPDH in each condition .

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Western blot showing the protein level of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.


Left : Representative live - imaging of EGFP - EB1 in control , CAMSAP2 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . Scale bar : 20 μm . Right : of EB1 growth speed obtained by plusTipTracker . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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Left: Representative live-cell fluorescence imaging of EGFP-EB1 in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of EB1 growth speed obtained by plusTipTracker. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Western blot showing level of active RhoA , total RhoA in negative control CAMSAP2 siRNA transfected hRPEs . GAPDH is used as loading control . Bar chart shows of normalized to GAPDH in each condition .

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Western blot showing the protein level of active RhoA, total RhoA in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.


Left : Representative live - imaging of GFP - AHPH in negative control CAMSAP2 siRNA transfected hRPEs . Scale bar : 20 μm . Right : of GFP - AHPH .

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Left: Representative live-cell fluorescence imaging of GFP-AHPH in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of GFP-AHPH fluorescence intensity.


Left : Representative immunofluorescent of pMLC ( yellow ) DAPI ( blue ) in negative control CAMSAP2 siRNA transfected hRPEs . Scale bar : 20 μm . Right : of pMLC . Below : Western blot showing level of pMLC in negative control CAMSAP2 siRNA transfected hRPEs . α - tubulin is used as loading

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Left: Representative immunofluorescent staining of pMLC (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of pMLC fluorescence intensity. Below: Western blot showing the protein level of pMLC in negative control and CAMSAP2 siRNA transfected hRPEs. α-tubulin is used as loading control


Left : showing collagen gel deformation at 0 h , 8 h 24 h . HRPEs transfected with negative control , CAMSAP2 AKAP450 siRNA were seeded in 1 % collagen gel ( yellow dashed lines ) . Scale bar : 5 mm . Right : of gel .

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Left: Images showing collagen gel deformation at 0 h, 8 h and 24 h. HRPEs transfected with negative control, CAMSAP2 and AKAP450 siRNA were seeded in 1% collagen gel (yellow dashed lines). Scale bar: 5 mm. Right: Quantification of gel area.


Left : Representative live - imaging of RFP - paxillin in control nocodazole ( 8 μM , 30 min ) treated hRPEs . Scale bar : 20 μm . Right : of FA obtained by FAAS .

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Left: Representative live-cell fluorescence imaging of RFP-paxillin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Quantification of FA area obtained by FAAS.


Top : Representative live - imaging of RFP - paxillin in negative control CAMSAP2 siRNA transfected hRPEs . Time - lapse imaging zoomed from yellow box showed assembly disassembly stage of a single FA . Scale bar : 20 μm . Bottom : of FA , assembly rate disassembly rate obtained by FAAS . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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Top: Representative live-cell fluorescence imaging of RFP-paxillin in negative control and CAMSAP2 siRNA transfected hRPEs. Time-lapse fluorescence imaging zoomed from the yellow box showed the assembly and disassembly stage of a single FA. Scale bar: 20 μm. Bottom: Quantification of FA area, assembly rate and disassembly rate obtained by FAAS. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Top : Schematic diagram of FA - MT contact . Position of EB1 paxillin were extracted from live - , followed by automated co - localization identification . Scale bar : 20 μm . Bottom : Representative time - lapse imaging of EGFP - EB1 RFP - paxillin zoomed from yellow box above showed targeting of MT to FA . FA - MT contact was calculated by of co - localization events to total EB1 number . FA - MT contact of negative control , CAMSAP2 , AKAP450 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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Top: Schematic diagram of FA-MT contact analysis. Position of EB1 and paxillin were extracted from live-cell images, followed by automated co-localization identification. Scale bar: 20 μm. Bottom: Representative time-lapse fluorescence imaging of EGFP-EB1 and RFP-paxillin zoomed from the yellow box above showed the targeting of MT to FA. FA-MT contact ratio was calculated by the ratio of co-localization events to the total EB1 number.   FA-MT contact ratio of negative control, CAMSAP2, AKAP450 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Western blot showing level of ELKS in negative control ELKS siRNA transfected hRPEs . α - tubulin was used as loading control . Bar chart shows of normalized to α - tubulin in each condition .

Western blot showing the protein level of ELKS in negative control and ELKS siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition.


FA - MT contact of negative control ELKS siRNA transfected hRPEs . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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FA-MT contact ratio of negative control and ELKS siRNA transfected hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Y FMI of negative control ELKS siRNA transfected hRPEs moving on stiffness gradient gels .

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Y FMI of negative control and ELKS siRNA transfected hRPEs moving on stiffness gradient gels.


Left : Representative immunofluorescent of GM130 ( yellow ) , F - actin ( gray ) DAPI ( blue ) in negative control CAMSAP2 siRNA transfected hRPEs at edge of wound after 8 h . Scale bar : 50 μm . Middle : Schematic diagram of Golgi - nucleus orientation towards front ( 45 ° - 135 ° ) or rear ( - 45 ° - - 135 ° ) . Right : of of Golgi - nucleus orientation towards front or rear of .

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Left: Representative immunofluorescent staining of GM130 (yellow), F-actin (gray) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h. Scale bar: 50 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the front (45°-135°) or the rear (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the front or rear of the cell.


Left : Representative immunofluorescent of GM130 ( yellow ) DAPI ( blue ) in negative control CAMSAP2 siRNA transfected hRPEs on stiffness gradient gel . Scale bar : 20 μm . Middle : Schematic diagram of Golgi - nucleus orientation towards stiff side ( 45 ° - 135 ° ) or soft side ( - 45 ° - - 135 ° ) . Right : of of Golgi - nucleus orientation towards stiff or soft side .

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Left: Representative immunofluorescent staining of GM130 (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs on the stiffness gradient gel. Scale bar: 20 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the stiff side (45°-135°) or the soft side (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the stiff or soft side.


Left : Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm irradiation . Middle : Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope . Right : Representative live - imaging of marked with Golgi ( green ) nucleus ( blue ) on soft region stiff island .

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Left: Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm laser irradiation. Middle: Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope. Right: Representative live-cell fluorescence imaging of cells marked with the Golgi (green) and nucleus (blue) on the soft region and the stiff island.


Representative time - lapse showing migration of hRPE to stiff island reorientation of Golgi in this process . Scale bar : 20 μm . of of having a stiff - end biased Golgi positioning . N = 29 , replicates are biological .

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Representative time-lapse fluorescence images showing the migration of hRPE cell to the stiff island and the reorientation of Golgi in this process. Scale bar: 20 μm.   Quantification of the percentage of cells having a stiff-end biased Golgi positioning. N=29 cells, replicates are biological.


b , MEFs were generated from day 12 . 5embryos of Atg16l1 mutant mice , western blot was used to detect a b isoforms of ATG16L1 from whole -

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b, MEFs were generated from day 12.5embryos of Atg16l1 mutant mice, and western blot analysis was used to detect the a and b isoforms of ATG16L1 from whole-cell lysates


c , d , Atg5 - / - ATG16L1HM MEFs were grown in presence of chemical inducer of autophagy rapamycin ( Rap , 50 µg ml - 1 ) cyclohexamide ( CHX , 5 µg ml - 1 ) for 0 , 4 , 8 12 h . lysates were analysed by western blot for loss of P62 expression ( c ) . P62 were quantified by densitometry normalized to actin ( n = 3 ; d ) . * P 0 . 04 ; * * P 0 . 008 ; * * * P = 0 .

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c, d, Atg5-/- and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 µg ml-1) and cyclohexamide (CHX, 5 µg ml-1) for 0, 4, 8 and 12 h. Cell lysates were analysed by western blot for loss of P62 expression (c). P62 levels were quantified by densitometry normalized to actin (n = 3; d). *P 0.04; **P 0.008; ***P = 0.0003


e , f , Western blot of LC3 expression in MEFs grown in presence of rapamycin ( 50 µg ml - 1 ) or dimethylsulphoxide ( DMSO ) control for 4 h ( e ) . LC3 - II expression in presence of rapamycin was quantified normalized to actin ( n = 6 ; f ) .

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e, f, Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin (50 µg ml-1) or dimethylsulphoxide (DMSO) control for 4 h (e). LC3-II expression in the presence of rapamycin was quantified and normalized to actin (n = 6; f).


g , Detection of ATG16L1 , LC3 P62 by western blot of ileal lysates from ATG16L1HM mice . ATG16L1 can be detected in both mutant lines on longer exposures . h , i , of ATG16L1 ( h ) P62 ( i ) normalized to actin detected in ileal lysates ( n = 3 ) . P values were calculated using two - Student ' s t - . Error bars , s . e . m .

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g, Detection of ATG16L1, LC3 and P62 by western blot of ileal lysates from ATG16L1HM mice. ATG16L1 can be detected in both mutant lines on longer exposures. h, i, Quantification of ATG16L1 (h) and P62 (i) normalized to actin detected in ileal lysates (n = 3). P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


a , b , Whole - mount taken immediately above ileal mucosal surface from WT ( a ) ATG16L1HM ( b ) littermate mice stained with Helix pomatia lectin that labels mucus ( green ) antisera directed against lysozyme ( red ) . are representative of 3 WT 3 ATG16L1HM mice .

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a, b, Whole-mount images taken immediately above the ileal mucosal surface from WT (a) and ATG16L1HM (b) littermate mice stained with Helix pomatia lectin that labels mucus (green) and antisera directed against lysozyme (red). Images are representative of 3 WT and 3 ATG16L1HM mice.


c , d , Ileal sections from WT ( c ) ATG16L1HM ( d ) mice were stained with PAS / alcian blue to detect Paneth granules by microscopy ( dashed denotes crypt unit arrowheads indicate Paneth ) . are representative of 7 WT , 7 ATG16L1HM1 5 ATG16L1HM2 mice ; more than 100 crypts were analysed for

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c, d, Ileal sections from WT (c) and ATG16L1HM (d) mice were stained with PAS/alcian blue to detect Paneth granules by light microscopy (dashed line denotes the crypt unit and arrowheads indicate Paneth cells). Images are representative of 7 WT, 7 ATG16L1HM1 and 5 ATG16L1HM2 mice; more than 100 crypts were analysed for each


e , f , Representative of indirect immunofluorescence of sections stained for lysozyme ( red ) in WT ( e ) ATG16L1HM ( f ) mouse ileal crypts .

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e, f, Representative images of indirect immunofluorescence of sections stained for lysozyme (red) in WT (e) and ATG16L1HM (f) mouse ileal crypts.


g , Paneth display one of four patterns of lysozyme expression ( represented in red ; represents areas that exclude lysozyme ) : normal ( D0 ) , disordered ( D1 ) , depleted ( D2 ) diffuse ( D3 ) .

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g, Paneth cells display one of four patterns of lysozyme expression (represented in red; white represents areas that exclude lysozyme): normal (D0), disordered (D1), depleted (D2) and diffuse (D3).


h , i , Number of Paneth from ATG16L1HM ( h ) Atg5flox / floxvillin - Cre ( i ) mice displaying each pattern of lysozyme expression ( n = 6 , 660 from 5 ATG16L1HM mice 5 , 634 from 5 WT mice ; n = 1 , 756 from 2 Atg5flox / floxvillin - Cre mice 1 , 649 from 2 Atg5flox / flox mice ) . Scale bars : a , b , 200 µm ; c - f , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . P values were calculated using two - Student ' s t - . Error bars , s . e . m .

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h, i, Number of Paneth cells from ATG16L1HM (h) and Atg5flox/floxvillin-Cre (i) mice displaying each pattern of lysozyme expression (n = 6,660 cells from 5 ATG16L1HM mice and 5,634 cells from 5 WT mice; n = 1,756 cells from 2 Atg5flox/floxvillin-Cre mice and 1,649 from 2 Atg5flox/flox mice). Scale bars: a, b, 200 µm; c-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


a - d , Electron micrograph of littermate WT ( a , c ) ATG16L1HM ( b , d ) Paneth ( for a , b , dashed lines denote , asterisk indicates normal progenitor ; for c , d , blue stars indicate normal mitochondria in WT Paneth ATG16L1HM progenitor , red diamonds indicate degenerating mitochondria , dashed box shows normal Golgi compartments , yellow asterisks indicate granules ; n = 3 mice per genotype ) . e , Mitochondrial integrity was quantified on basis of of visible mitochondrial section displaying intact cisternae ( n = 49 WT 71 ATG16L1HM ) .

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a-d, Electron micrograph of littermate WT (a, c) and ATG16L1HM (b, d) Paneth cells (for a, b, dashed lines denote individual cells, asterisk indicates normal progenitor cell; for c, d, blue stars indicate normal mitochondria in WT Paneth cells and ATG16L1HM progenitor cells, red diamonds indicate degenerating mitochondria, the dashed box shows normal Golgi compartments, and yellow asterisks indicate granules; n = 3 mice per genotype). e, Mitochondrial integrity was quantified on the basis of the percentage of the visible mitochondrial section displaying intact cisternae (n = 49 WT and 71 ATG16L1HM cells).


g , Chart of selected genes for which mRNA is enriched in either ATG16L1 - deficient Paneth ( baseline is WT Paneth ) or ATG16L1 - deficient thymocytes ( baseline is WT thymocytes ) . Only haemoglobin is in common in these two sets of analyses . NS , not significant . Scale bars : a , b , 10 µm ; c , d , 500 nm .

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g, Chart of selected genes for which mRNA is enriched in either ATG16L1-deficient Paneth cells (baseline is WT Paneth cells) or ATG16L1-deficient thymocytes (baseline is WT thymocytes). Only haemoglobin is in common in these two sets of analyses. NS, not significant. Scale bars: a, b, 10 µm; c, d, 500 nm.


a , b , Haematoxylin - - eosin - stained sections of uninvolved areas from ileo - colic resections from with Crohn ' s disease homozygous for safe ( a ) or risk ( b ) allele of ATG16L1 ( blue dashed denotes crypt unit ) .

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a, b, Haematoxylin-and-eosin-stained sections of uninvolved areas from ileo-colic resections from patients with Crohn's disease homozygous for the safe (a) or risk (b) allele of ATG16L1 (blue dashed line denotes crypt unit).


c - f , Immunofluorescence of Paneth from control ( c , e ) with risk allele ( d , f ) stained for lysozyme ( red ; c , d ) double - labelled additionally for leptin ( green ; e , f ; yellow dashed denotes crypt unit ) .

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c-f, Immunofluorescence images of Paneth cells from control patients (c, e) and patients with the risk allele (d, f) stained for lysozyme (red; c, d) and double-labelled additionally for leptin (green; e, f; yellow dashed line denotes the crypt unit).


g , Aberrant lysozyme expression was quantified using same criteria as that used for mouse sections in Fig . 3 ( n = 6 , 829 for at risk 8 , 182 for control , n = 5 for each genotype ) . Leptin - positive D3 were quantified in samples ( n = 4 per genotype ) homozygous for risk allele ( 76 out of 322 D3 were positive from a total of 580 crypts examined ) homozygous for safe allele ( 11 out of 93 D3 were positive from a total 749 crypts examined ) . Scale bars : a - f , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . P values were calculated using two - Student ' s t - . Error bars , s . e . m .

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g, Aberrant lysozyme expression was quantified using the same criteria as that used for mouse sections in Fig. 3 (n = 6,829 cells for at risk patients and 8,182 for control, n = 5 patients for each genotype). Leptin-positive D3 cells were quantified in patient samples (n = 4 patients per genotype) homozygous for the risk allele (76 out of 322 D3 cells were positive from a total of 580 crypts examined) and homozygous for the safe allele (11 out of 93 D3 cells were positive from a total 749 crypts examined). Scale bars: a-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


MIP of cerebral vessels of 3dpf Tg ( kdrl : HRAS - mCherry ) s916 ( grey LUT ; inverted ) . Higher magnification panel showing two kugeln ( arrowheads ) arising from middle mesencephalic central artery ( MMCtA ) .

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MIP of cerebral vessels of 3dpf Tg(kdrl:HRAS-mCherry)s916 embryo (grey LUT; inverted). Higher magnification panel showing two kugeln (arrowheads) arising from the middle mesencephalic central artery (MMCtA).


Diameter of kugeln at 3dpf ( mean ± s . e . m . 10 . 13 ± 0 . 49 ; n = 93 kugeln from 32 3dpf ; 3 experimental repeats ) .

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Diameter of kugeln at 3dpf (mean ± s.e.m. 10.13 ± 0.49; n=93 kugeln from 32 3dpf embryos; 3 experimental repeats).


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Number of kugeln per embryo in 3, 4 and 5dpf animals was not statistically significantly different (mean ± s.e.m. 3dpf 5.47 ± 1.09, 4dpf 7.47 ± 1.46, 5dpf 7.09 ± 1.55; p = 0.8571; 3dpf: 175 kugeln from 32 embryos; 4dpf: 239 kugeln from 32 embryos, 5dpf: 227 kugeln from 32 embryos; 4 experimental repeats; Kruskal-Wallis test).


Kugeln ( arrowheads ) could be observed in 28dpf .

Kugeln (arrowheads) could be observed in 28dpf animals.


MIP of cerebral vessels of 4dpf Tg ( fli1aep : CAAX - eGFP ) ( grey LUT ; inverted ) . Higher magnification panel shows a kugel ( arrowhead ) protruding from posterior mesencephalic central artery ( PMCtA ) .

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MIP of cerebral vessels of 4dpf Tg(fli1aep:CAAX-eGFP) embryo (grey LUT; inverted). Higher magnification panel shows a kugel (arrowhead) protruding from the posterior mesencephalic central artery (PMCtA).


Single Z - plane micrograph of cerebral vessels of a 3dpf Tg ( gata1 : dsRed ) , injected with a Tol2 - fli1a : myr - Cherry construct showing two kugeln ( arrowheads ) in higher magnification panel protruding from MMCtA .

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Single Z-plane micrograph of cerebral vessels of a 3dpf Tg(gata1:dsRed), injected with a Tol2-fli1a:myr-Cherry construct showing two kugeln (arrowheads) in the higher magnification panel protruding from the MMCtA.


Locations proportion of kugeln on cerebral vessels ( colour coded by vessel ; total n = 107 kugeln from 34 4dpf ; 3 experimental repeats ) . AMCtA - anterior mesencephalic central artery , BCA - basal communicating artery , CaDI - caudal division of internal carotid artery , CMV - communicating vessel , CtA - central arteries , MMCtA - middle mesencephalic central artery , MtA - metencephalic artery , PCS - posterior communicating segment , PMBC - posterior midbrain channel , PMCtA - posterior mesencephalic central artery , PrA - prosencephalic artery .

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Locations and proportion of kugeln on cerebral vessels (colour coded by vessel; total n=107 kugeln from 34 4dpf embryos; 3 experimental repeats). AMCtA - anterior mesencephalic central artery, BCA - basal communicating artery, CaDI - caudal division of internal carotid artery, CMV - communicating vessel, CtA - central arteries, MMCtA - middle mesencephalic central artery, MtA - metencephalic artery, PCS - posterior communicating segment, PMBC - posterior midbrain channel, PMCtA - posterior mesencephalic central artery, PrA - prosencephalic artery.


MIPs of a time - series sheet acquisition shows three different kugeln ( kugel 1 - black arrowhead ; kugel 2 - grey arrowhead ; kugel 3 - unfilled arrowhead ) protruding retracting from parent vessels ( 2min intervals ; inverted LUT ) .

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MIPs of a time-series light sheet acquisition shows three different kugeln (kugel 1 - black arrowhead; kugel 2 - grey arrowhead; kugel 3 - unfilled arrowhead) protruding and retracting from parent vessels (2min intervals; inverted LUT).


Kugeln persisted for variable durations before regression into parent vessel ( 43 kugeln from 9 4dpf embyros ; 4 experimental repeats ) .

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Kugeln persisted for variable durations before regression into the parent vessel (43 kugeln from 9 4dpf embyros; 4 experimental repeats).


MIPs taken 20min apart showing examples of different kugel behaviour including shape changes ( asterisk ) , expansion ( circle ) , retraction ( triangle ) , or little change ( pentagon ) .

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MIPs taken 20min apart showing examples of different kugel behaviour including shape changes (asterisk), expansion (circle), retraction (triangle), or little change (pentagon).


MIPs taken 1h apart show that kugeln may be observed at one time - point ( red arrowhead , 55 : 20 : 00 ) but not 1h later in same ( 56 : 20 : 00 ) to develop on other vessels 1h later ( 57 : 20 : 00 ) which again have regressed by 58 : 20 : 00 .

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MIPs taken 1h apart show that kugeln may be observed at one time-point (red arrowhead, 55:20:00) but not 1h later in the same animal (56:20:00) and to develop on other vessels 1h later (57:20:00) which again have regressed by 58:20:00.


Kymographs generated by - scanning across diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes , while no such oscillations were observed in adjacent similarly sized cerebral vessels ( grey LUT ; inverted ) .

Kymographs generated by line-scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes, while no such oscillations were observed in adjacent similarly sized cerebral vessels (all images grey LUT; inverted).


Double transgenic visualizing endothelial ( red ) endothelial nuclei ( green ) . Endothelial nuclei ( arrowhead ) were observed close to but never within kugel ( unfilled arrowhead ) .

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Double transgenic visualizing endothelial membrane (red) and endothelial nuclei (green). Endothelial nuclei (arrowhead) were observed close to but never within the kugel (unfilled arrowhead).


Double transgenic showing endothelial ( red ) endothelial F - actin ( green ) . F - actin was found to localize at neck of

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Double transgenic showing endothelial membrane (red) and endothelial F-actin (green). F-actin was found to localize at the neck of the kugeln


Double transgenic showing endothelial ( red ) endothelial cytoplasm ( green ) . reporter was visible in parent vessel but not in kugel ( unfilled arrowhead ) .

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Double transgenic showing endothelial membrane (red) and endothelial cytoplasm (green). The cytoplasmic reporter was visible in the parent vessel but not in the kugel (unfilled arrowhead).


Triple transgenic showing endothelial ( red ) , neurons ( green ) erythrocytes ( red ) . Surrounding neurons were excluded from volume of kugel ( unfilled arrowhead ) erythrocytes were not observed inside kugeln ( arrowhead ) .

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Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead) and erythrocytes were not observed inside kugeln (arrowhead).


Treatment with inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per ( 100nM 1 hour ; * * p = 0 . 0041 ; control n = 21 8 . 05 ± 1 . 76 ( mean ± s . e . m . ) ; Latrunculin n = 21 17 . 19 ± 2 . 93 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100nM 1 hour; **p = 0.0041; control n=21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n=21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Latrunculin B treatment statistically significantly reduced kugel diameter ( * p = 0 . 0164 ; control n = 169 kugeln from 21 7 . 56 ± 2 . 25 ( mean ± s . e . m . ) ; Latrunculin n = 361 kugeln from 21 5 . 91 ± 1 . 78 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - ) .

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Latrunculin B treatment statistically significantly reduced kugel diameter (*p = 0.0164; control n=169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n=361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).


Treatment with Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per ( 25 µM 1h ; * * * * p < 0 . 0001 ; control n = 22 3 . 77 ± 0 . 56 ( mean ± s . e . m . ) , Blebbistatin n = 24 1 . 08 ± 0 . 22 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 µM 1h; ****p <0.0001; control n=22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n=24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Blebbistatin treatment had no effect on kugel diameter ( p = 0 . 3731 ; control n = 83 kugeln from 22 6 . 27 ± 0 . 72 ( mean ± s . e . m . ) , Blebbistatin n = 26 kugeln from 24 7 . 02 ± 0 . 89 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Blebbistatin treatment had no effect on kugel diameter (p = 0.3731; control n=83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n=26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


MIP of cerebral vessels of a 4dpf before exsanguination ( grey LUT ; inverted ) . MIP of same after exsanguination , which did not alter kugel .

MIP of the cerebral vessels of a 4dpf embryo before exsanguination (grey LUT; inverted). MIP of the same embryo after exsanguination, which did not alter kugel size.


Time - lapse imaging of an with transiently halted cardiac contraction ( using Tricaine ) . Despite absent blood flow kugeln still changed shape ( grey arrowhead ) , retained shape ( arrowhead ) or protruded retracted ( black arrowhead ; time post cessation of flow is indicated on ; 3dpf ; grey inverted LUT ) . Time - lapse of an with transiently halted cardiac contraction as in ( C ) showed that kugel diameter still oscillates ( time post cessation of flow ) .

Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow).


Dextran microangiography filled perfused vessels with dextran ( arrowhead ) , while dextran was not observed to enter kugeln ( unfilled arrowhead ) .

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

Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead).


Inhibition of cardiac contraction by tnnt2a morpholino ( MO ) knockdown statistically significantly reduced kugel number per ( * * * * p < 0 . 0001 ; control n = 20 5 . 10 ± 1 . 47 ( mean ± s . e . m . ) , tnnt2a MO = 18 0 . 06 ± 0 . 06 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugeln were studied in double - transgenic Tg ( fli1a : LifeAct - mClover ) sh467 , Tg ( kdrl : HRAS - mCherry ) s916 . BLECs were mClover positive mCherry - negative ( BLECs - black arrowheads ; kugeln - arrowheads ) .

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

Kugeln were studied in the double-transgenic Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916. BLECs were mClover positive and mCherry-negative (BLECs - black arrowheads; kugeln - white arrowheads).


No direct physical interaction between kugeln ( BLECs - black arrowheads ; kugeln - arrowheads ) BLECs was observed ( n = 21 4dpf ; 3 experimental repeats ) . Depth - coded MIPs showed that BLECs kugeln are present on different anatomical planes ( depths ; purple ventral ( v ) , dorsal ( d ) ; BLECs - black arrowheads ; kugeln - arrowheads ) .

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

No direct physical interaction between kugeln (BLECs - black arrowheads; kugeln - white arrowheads) and BLECs was observed (n=21 4dpf embryos; 3 experimental repeats). Depth-coded MIPs showed that BLECs and kugeln are present on different anatomical planes (depths; purple ventral (v), white dorsal (d); BLECs - black arrowheads; kugeln - white arrowheads).


Ccbe1 morpholino ( MO ) injection led to a loss of lymphatics ( arrowhead ) .

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

Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead).


Kugel number was not statistically significantly altered by ccbe1 morpholino ( MO ) knockdown ( p = 0 . 3496 ; control MO n = 22 0 . 95 ± 0 . 28 ( mean ± s . e . m . ) , ccbe1 MO n = 23 0 . 57 ± 0 . 14 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown ( p = 0 . 8783 ; control MO n = 21 kugeln from 22 7 . 75 ± 1 . 29 ( mean ± s . e . m . ) , ccbe1 MO n = 13 kugeln from 23 8 . 93 ± 2 . 35 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Injection of IgG - conjugated Alexa 647 into tectum showed no uptake ( blue ; unfilled arrowhead ) by kugeln ( arrowhead ; 140 kugeln from 17 4dpf ; 2 experimental repeats ) .

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

Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).


possible interaction of macrophages with kugeln was studied in transgenic Tg ( kdrl : HRAS - mCherry ) s916 , Tg ( fms : GAL4 . VP16 ) i186 , Tg ( UAS - E1b : nfsB . mCherry ) il149 , visualizing macrophages vasculature simultaneously . Coloured depth - coding indicates different anatomical depths of kugeln macrophages ( ventral purple , dorsal ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously. Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white).


Maximum projections of z - stacks acquired every 10 minutes during a time - lapse showed no interaction of macrophages ( unfilled arrowhead ) with kugeln ( filled arrowhead ; n = 21 kugeln in 8 3dpf ) . Macrophages are , indicating they are on a different z - plane to vessels seen .

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

Maximum intensity projections of z-stacks acquired every 10 minutes during a time-lapse showed no interaction of macrophages (unfilled arrowhead) with kugeln (filled arrowhead; n=21 kugeln in 8 3dpf embryos). Macrophages are all white, indicating they are on a different z-plane to the vessels seen.


VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number ( * * * * p < 0 . 0001 ; DMSO control n = 30 8 . 53 ± 2 . 62 ( mean ± s . e . m . ) ; AV951 n = 31 21 . 10 ± 2 . 71 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Mann - Whitney U ) .

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

VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number (****p<0.0001; DMSO control n=30 embryos 8.53 ± 2.62 (mean ± s.e.m.); AV951 n=31 embryos 21.10 ± 2.71 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly different after AV951 treatment ( p = 0 . 7890 ; DMSO control n = 243 kugeln from 30 9 . 94 ± 0 . 76 ( mean ± s . e . m . ) ; AV951 n = 652 kugeln from 31 9 . 73 ± 0 . 32 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; 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

Mean kugel diameter was not statistically significantly different after AV951 treatment (p = 0.7890; DMSO control n=243 kugeln from 30 embryos 9.94 ± 0.76 (mean ± s.e.m.); AV951 n=652 kugeln from 31 embryos 9.73 ± 0.32 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Student's t-test).


DAF - FM , a vital dye for nitric oxide ( NO ) , showed that 57 . 56 % of kugeln were positive for nitric oxide reactivity ( 2 . 5µM incubation from 96 - 102hpf ; n = 22 4dpf ; 118 of 205 kugeln filled ; 3 experimental repeats ) . show representative " filled " ( DAF - FM positive ) " unfilled " ( DAF - FM negative ) kugeln .

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

DAF-FM staining, a vital dye for nitric oxide (NO), showed that 57.56% of kugeln were positive for nitric oxide reactivity (2.5µM incubation from 96-102hpf; n=22 4dpf embryos; 118 of 205 kugeln filled; 3 experimental repeats). Images show representative "filled" (DAF-FM positive) and "unfilled" (DAF-FM negative) kugeln.


Time - lapse acquisition with DAF - FM revealed that kugeln contained NO early in their biogenesis ( grey LUT ; inverted ) .

Time-lapse acquisition with DAF-FM revealed that kugeln contained NO early in their biogenesis (grey LUT; inverted).


Application of LysoTracker , a vital dye that stains lysosomes or acidic compartments , showed that 17 . 08 % of kugeln contained acidic contents ( 8 . 33µM ; 96 - 101hpf ; n = 22 4dpf ; 62 of 363 kugeln filled ; 3 experimental repeats ) . show representative " filled " ( Lysotracker positive ) " unfilled " ( Lysotracker negative ) kugeln .

Application of LysoTracker, a vital dye that stains lysosomes or acidic compartments, showed that 17.08% of kugeln contained acidic contents (8.33µM; 96-101hpf; n=22 4dpf embryos; 62 of 363 kugeln filled; 3 experimental repeats). Images show representative "filled" (Lysotracker positive) and "unfilled" (Lysotracker negative) kugeln.


nitric oxide synthase ( NOS ) inhibitor L - NAME had no statistically significant effect on kugel number per ( 0 . 5mM L - NAME 18h ; p = 0 . 4870 ; control n = 22 14 . 27 ± 3 . 35 ( mean ± s . e . m . ) , L - NAME n = 24 7 . 46 ± 1 . 61 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

The nitric oxide synthase (NOS) inhibitor L-NAME had no statistically significant effect on kugel number per embryo (0.5mM L-NAME 18h; p = 0.4870; control n=22 embryos 14.27 ± 3.35 (mean ± s.e.m.), L-NAME n=24 embryos 7.46 ± 1.61 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Diameter of kugeln was not affected by L - NAME ( p = 0 . 4161 ; control n = 315 kugeln from 22 8 . 77 ± 0 . 49 ( mean ± s . e . m . ) , L - NAME n = 179 kugeln from 24 8 . 66 ± 0 . 71 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Diameter of kugeln was not affected by L-NAME (p = 0.4161; control n=315 kugeln from 22 embryos 8.77 ± 0.49 (mean ± s.e.m.), L-NAME n=179 kugeln from 24 8.66 ± 0.71 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT ( * * * * p < 0 . 0001 ; control n = 24 9 . 38 ± 1 . 77 ( mean ± s . e . m . ) , DAPT n = 24 0 . 92 ± 0 . 28 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly altered by DAPT treatment ( p = 0 . 0832 ; control n = 225 kugeln from 24 8 . 64 ± 0 . 54 ( mean ± s . e . m . ) ; DAPT n = 22 kugeln from 24 6 . 88 ± 0 . 86 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - ) .

Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).


A transgenic dll4 reporter showed no indication of different dll4 expression at sites of kugeln ( arrowheads pointing to kugeln ; n = 122 kugeln from 23 3dpf ; 2 experimental repeats ) .

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

A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln (white arrowheads pointing to kugeln; n=122 kugeln from 23 3dpf embryos; 2 experimental repeats).


Studying Notch signalling in Notch reporter Tg ( TP1glob : venusPest ) s940 , showed high Notch in mid - brain , but , again , no difference in expression at sites of kugeln ( n = 45 kugeln from 19 4dpf ; 2 experimental repeats ) .

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

Studying Notch signalling in the Notch reporter line Tg(TP1glob:venusPest)s940, showed high Notch levels in the mid-brain, but, again, no difference in expression at the sites of kugeln (n=45 kugeln from 19 4dpf embryos; 2 experimental repeats).


Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV - 939 ( * * * p = 0 . 0003 ; control n = 22 1 . 32 ± 0 . 24 ( mean ± s . e . m . ) ; XAV - 939 n = 21 4 . 29 ± 0 . 71 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by XAV - 939 ( p = 0 . 4098 ; control n = 29 kugeln from 22 8 . 78 ± 0 . 76 ( mean ± s . e . m . ) ; XAV - 939 n = 90 kugeln from 21 7 . 99 ± 0 . 56 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Student ' s t - )

Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test)


Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV ( p 0 . 0359 ; control n = 22 1 . 04 ± 0 . 34 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 21 4 . 24 ± 1 . 28 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


diameter of kugel was not statistically significantly altered by GSK3 inhibition ( p 0 . 5555 ; control n = 23 kugeln from 22 7 . 26 ± 0 . 89 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 89 kugeln from 21 8 . 09 ± 0 .

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

The diameter of kugel was not statistically significantly altered by GSK3 inhibition (p 0.5555; control n=23 kugeln from 22 embryos 7.26 ± 0.89 (mean ± s.e.m.); GSK3 inhibitor n=89 kugeln from 21 embryos 8.09 ± 0.97


( C ) Co - immunoprecipitation between VP30 indicated PPxPxY containing host factors . Empty expression plasmid ( Vector ) or FLAG - tagged host expression plasmids were co - transfected with an HA - VP30 expression plasmid in HEK293T . An anti - FLAG immunoprecipitation ( IP : Anti - FLAG ) was performed . Western blots of IP whole lysates ( WCL ) are shown . Anti - β - tubulin was used as a loading control for WCLs .

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

(C) Co-immunoprecipitation between VP30 and the indicated PPxPxY containing host factors. Empty expression plasmid (Vector) or FLAG-tagged host protein expression plasmids were co-transfected with an HA-VP30 expression plasmid in HEK293T cells. An anti-FLAG immunoprecipitation (IP: Anti-FLAG) was performed. Western blots of IP and whole cell lysates (WCL) are shown. Anti-β-tubulin was used as a loading control for the WCLs.


( D ) were transfected with empty vector or HA - VP30 expression plasmid IPs were performed with anti - HA antibody ( IP : Anti - HA ) . Western blotting with anti - HA tag or antibodies to indicated host was performed on IP on whole lysates ( WCL ) .

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

(D) Cells were transfected with empty vector or HA-VP30 expression plasmid and IPs were performed with anti-HA antibody (IP: Anti-HA). Western blotting with anti-HA tag or antibodies to the indicated host proteins was performed on IP and on whole cell lysates (WCL).


( B ) Co - immunoprecipitation between HA - VP30 either GFP or GFP - fused to peptides derived from host was performed . Western blots of co - immunoprecipitation whole - lysate ( WCL ) are shown .

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

(B) Co-immunoprecipitation between HA-VP30 and either GFP or GFP-fused to peptides derived from the host proteins was performed. Western blots of the co-immunoprecipitation and whole-cell lysate (WCL) are shown.


( C ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 in presence of increasing concentrations ( 0 . 13 - 500 μM ) of PPxPxY containing peptides . polarization was determined with constant concentrations of FITC - RBBP6 eVP30130 - 272 , at 0 . 50 μM 3 . 8 μM respectively . Experiments were performed in two independent replicates . Error bars represent standard deviation .

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

(C) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 in the presence of increasing concentrations (0.13-500 μM) of the PPxPxY containing peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation.


( A ) HDX kinetics data for VP30 alone for indicated host peptides incubated with VP30 are shown . VP30 peptide acid residue positions sequences are indicated at top of each curve . error bars indicate mean ± S . D . ( N = 3 ) .

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

(A) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown. VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve. The error bars indicate mean ± S.D. (N=3).


( A ) Co - immunoprecipitation experiment demonstrating that NP interferes with VP30 - PPxPxY binding . VP30 was co - expressed with indicated HA - tagged PPxPxY either empty vector ( - ) or increasing amounts of FLAG - tagged NP expression plasmid . IPs were performed with anti - HA antibodies followed by Western blots of IPs or WCL by using anti - HA , anti - FLAG anti - VP30 antibodies .

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

(A) Co-immunoprecipitation experiment demonstrating that NP interferes with VP30-PPxPxY binding. VP30 was co-expressed with the indicated HA-tagged PPxPxY proteins and either empty vector (-) or increasing amounts of FLAG-tagged NP expression plasmid. IPs were performed with anti-HA antibodies followed by Western blots of IPs or WCL by using anti-HA, anti-FLAG and anti-VP30 antibodies.