( B ) Total lysates from HEK293T transfected with Flag - OFD1 were IP with anti - myosin VI antibodies ( 1295 and 1296 ) and an unrelated rabbit antibody ( anti - GST , as control ) . IB was performed with anti - Flag and anti - myosin VI antibodies . ( C ) Total lysates from HEK293T transfected with Flag - OFD1 or Flag ( as control ) were IP with anti - Flag antibody - conjugated beads . IB was performed with anti - Flag and anti - myosin VI antibodies .

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(B) Total lysates from HEK293T transfected with Flag-OFD1 were IP with anti-myosin VI antibodies (1295 and 1296) and an unrelated rabbit antibody (anti-GST, as control). IB was performed with anti-Flag and anti-myosin VI antibodies. (C) Total lysates from HEK293T transfected with Flag-OFD1 or Flag (as control) were IP with anti-Flag antibody-conjugated beads. IB was performed with anti-Flag and anti-myosin VI antibodies.


( D ) Selected sections deriving from TEM analysis of A549 wild - type versus myosin VI KO cells . The cells were immunogold - labelled with anti - myosin VI antibody . The centrioles in the images are indicated with white dashed circles . Scale bar , 200 nm . Top : representation of the estimated localisation of myosin VI at the centrioles .

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(D) Selected sections deriving from TEM analysis of A549 wild-type versus myosin VI KO cells. The cells were immunogold-labelled with anti-myosin VI antibody. The centrioles in the images are indicated with white dashed circles. Scale bar, 200 nm. Top: representation of the estimated localisation of myosin VI at the centrioles.


( F ) GST pulldown assay using myosin VI tail ( 835 - 1295 ) construct ( or GST alone as control ) and lysates from HEK293T cells transfected with the indicated Flag - OFD1 constructs ( or Flag alone , as control ) . IB was performed with anti - Flag antibody . Ponceau staining as indicated .

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(F) GST pulldown assay using myosin VI tail (835-1295) construct (or GST alone as control) and lysates from HEK293T cells transfected with the indicated Flag-OFD1 constructs (or Flag alone, as control). IB was performed with anti-Flag antibody. Ponceau staining as indicated.


D ) Quantification of OFD1 intensity at the mother or daughter centrioles . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mother centrioles : Mock , n = 148 cells ; MyoVI KD , n = 199 cells , from four independent experiments . Daughter centrioles : Mock , n = 101 cells ; MyoVI KD , n = 148 cells , from three independent experiments . * * * * P < 0 , 0001 by Mann - Whitney test .

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D) Quantification of OFD1 intensity at the mother or daughter centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n=148 cells; MyoVI KD, n=199 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; MyoVI KD, n=148 cells, from three independent experiments. **** P<0,0001 by Mann-Whitney test.


( F - G ) IF analysis of Cep164 signal . hTERT - RPE1 cells were transfected with siRNA against myosin VI and / or OFD1 . Four days after transfection , cells were treated with nocodazole ( 1 hour , 6 μg / ml ) and immunostained with anti - OFD1 , anti - centrin1 and anti - Cep164 antibodies . ( F ) Representative images . Scale bar , 1 μm . ( G ) Quantification of Cep164 intensity at the mother centrioles . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock , n = 147 cells ; OFD1 KD , n = 146 cells ; MyoVI KD , n = 150 cells ; MyoVI + OFD1 KD , n = 151 cells , from four independent experiments . * * P < 0 , 005 ; * * * * P < 0 , 0001 by Kruskal - Wallis test .

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(F-G) IF analysis of Cep164 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. (F) Representative images. Scale bar, 1 μm. (G) Quantification of Cep164 intensity at the mother centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=147 cells; OFD1 KD, n=146 cells; MyoVI KD, n=150 cells; MyoVI + OFD1 KD, n=151 cells, from four independent experiments. ** P<0,005; **** P<0,0001 by Kruskal-Wallis test.


( A ) Growth curve of hTERT - RPE1 cells transfected with myosin VI siRNA . A representative plot of three independent experiments is shown . ( B ) Growth curve of BJ - hTERT cells transfected with myosin VI siRNA . A representative plot of two independent experiments is shown .

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(A) Growth curve of hTERT-RPE1 cells transfected with myosin VI siRNA. A representative plot of three independent experiments is shown. (B) Growth curve of BJ-hTERT cells transfected with myosin VI siRNA. A representative plot of two independent experiments is shown.


( G ) Representative bright field images of cells treated with the indicated p53 and myosin VI siRNAs . Scale bar , 200 μm .

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(G) Representative bright field images of cells treated with the indicated p53 and myosin VI siRNAs. Scale bar, 200 μm.


( I ) Quantification of OFD1 intensity at the mother or daughter centrioles . hTERT - RPE1 cells treated or not with Nutlin - 3 , were incubated with nocodazole for 1 hour ( 6 μg / ml ) and immunostained with anti - OFD1 , anti - centrin1 and anti - Cep164 antibodies . Mother centrioles were identified by the coincident staining of centrin1 and Cep164 , while daughter centrioles were centrin1 - only stained . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mother centrioles : Mock , n = 148 cells ; Nutlin - 3 , n = 169 cells , from four independent experiments . Daughter centrioles : Mock , n = 101 cells ; Nutlin - 3 , n = 122 cells , from three independent experiments . ns , not significant by Mann - Whitney test .

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(I) Quantification of OFD1 intensity at the mother or daughter centrioles. hTERT-RPE1 cells treated or not with Nutlin-3, were incubated with nocodazole for 1 hour (6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mother centrioles: Mock, n=148 cells; Nutlin-3, n=169 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; Nutlin-3, n=122 cells, from three independent experiments. ns, not significant by Mann-Whitney test.


( J ) Quantification of Cep164 intensity at the mother centrioles in hTERT - RPE1 cells treated as in ( I ) . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock , n = 195 cells ; Nutlin - 3 , n = 196 cells , from four independent experiments . ns , not significant by Mann - Whitney test .

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(J) Quantification of Cep164 intensity at the mother centrioles in hTERT-RPE1 cells treated as in (I). Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=195 cells; Nutlin-3, n=196 cells, from four independent experiments. ns, not significant by Mann-Whitney test.


( A ) Super - resolution analysis of OFD1 localisation at the centrioles . hTERT - RPE1 cells were transfected with siRNA against myosin VI , immunostained with anti - OFD1 and anti - acetylated tubulin antibodies and visualised using structured illumination microscopy ( SIM ) . Representative images are shown , scale bar , 500 nm . A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side .

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(A) Super-resolution analysis of OFD1 localisation at the centrioles. hTERT-RPE1 cells were transfected with siRNA against myosin VI, immunostained with anti-OFD1 and anti-acetylated tubulin antibodies and visualised using structured illumination microscopy (SIM). Representative images are shown, scale bar, 500 nm. A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side.


( C ) dSTORM super - resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI - depleted cells . To emphasise the symmetry of the structures , the signals from all nine appendages were averaged ( bottom ) . Representative images are shown ; scale bar , 200 nm .

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(C) dSTORM super-resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI-depleted cells. To emphasise the symmetry of the structures, the signals from all nine appendages were averaged (bottom). Representative images are shown; scale bar, 200 nm.


FRAP analysis of centriole - associated GFP - OFD1 . hTERT - RPE cells stably expressing GFP - OFD1 and centrin1 - dTomato were transfected with siRNA against myosin VI . After four days , cells were treated with nocodazole ( 1 hour , 6 μg / ml ) and subjected to live - cell imaging . ( E ) A representative graph of one out of three experiments . For each time point , the fraction of recovery of GFP - OFD1 is shown . Results are expressed as means with 95 % confidence interval . n = 12 cells ( Mock ) , n = 13 cells ( MyoVI KD ) . ( F ) Quantification of the half - time of fluorescence recovery ( t1 / 2 , ) and of the mobile fraction of GFP - OFD1 . Results are expressed as mean ± SD . n = 41 cells ( Mock ) , n = 31 cells ( MyoVI KD ) , from three independent experiments . ns , not significant ; * * * P < 0 , 0005 by Unpaired T - test .

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FRAP analysis of centriole-associated GFP-OFD1. hTERT-RPE cells stably expressing GFP-OFD1 and centrin1-dTomato were transfected with siRNA against myosin VI. After four days, cells were treated with nocodazole (1 hour, 6 μg/ml) and subjected to live-cell imaging. (E) A representative graph of one out of three experiments. For each time point, the fraction of recovery of GFP-OFD1 is shown. Results are expressed as means with 95% confidence interval. n=12 cells (Mock), n=13 cells (MyoVI KD). (F) Quantification of the half-time of fluorescence recovery (t1/2,) and of the mobile fraction of GFP-OFD1. Results are expressed as mean ±SD. n=41 cells (Mock), n=31 cells (MyoVI KD), from three independent experiments. ns, not significant; *** P<0,0005 by Unpaired T-test.


( B ) IF analysis of PCM1 signal . hTERT - RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti - PCM1 and anti - Cep135 antibodies . Upper panel , representative images , scale bar , 2 μm . Lower panel , quantification of PCM1 intensity . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock , n = 96 cell ; MyoVI KD , n = 98cells , from two independent experiments . * * * * P < 0 , 0001 by Mann - Whitney test . ( C ) IF analysis of PCM1 signal . hTERT - RPE1 cells were treated with Nutlin - 3 for 24 hours and immunostained with anti - PCM1 and anti - Cep135 antibodies . Panels as in B . Mock , n = 96 cells ; Nutlin - 3 , n = 100 cells , from two independent experiments . * * * * P < 0 , 0001 by Mann - Whitney test . ( D ) IF analysis of PCM1 signal . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti - PCM1 and anti - Cep164 antibodies . Panels as in B . Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole , identified with anti - Cep164 staining . Mock , n = 128 cells ; MyoVI KD , n = 114 cells from three independent experiments . ns , not significant by Mann - Whitney test .

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(B) IF analysis of PCM1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep135 antibodies. Upper panel, representative images, scale bar, 2 μm. Lower panel, quantification of PCM1 intensity. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=96 cell; MyoVI KD, n=98cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (C) IF analysis of PCM1 signal. hTERT-RPE1 cells were treated with Nutlin-3 for 24 hours and immunostained with anti-PCM1 and anti-Cep135 antibodies. Panels as in B. Mock, n=96 cells; Nutlin-3, n=100 cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (D) IF analysis of PCM1 signal. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep164 antibodies. Panels as in B. Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole, identified with anti-Cep164 staining. Mock, n=128 cells; MyoVI KD, n=114 cells from three independent experiments. ns, not significant by Mann-Whitney test.


( B ) IF analysis of OFD1 signal at the centriolar satellites upon serum starvation . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI . After four days , cells were fixed ( growing ) or serum starved for 24 hours ( SS ) . Cells were immunostained with anti - OFD1 and anti - PCM1 antibodies . The intensity of OFD1 signal in the area covered by PCM1 was quantified and normalised against the intensity of PCM1 staining in the same area . Left , representative images , scale bar , 2 μm . Right , results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock _ growing , n = 150 cells ; Mock _ SS , n = 149 cells ; MyoVI KD _ growing , n = 149 cells ; MyoVI KD _ SS , n = 150 cells , from three independent experiments . ns , not significant ; * P < 0 , 05 ; * * * * P < 0 , 0001 by Kruskal - Wallis test .

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(B) IF analysis of OFD1 signal at the centriolar satellites upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-OFD1 and anti-PCM1 antibodies. The intensity of OFD1 signal in the area covered by PCM1 was quantified and normalised against the intensity of PCM1 staining in the same area. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock_growing, n=150 cells; Mock_SS, n=149 cells; MyoVI KD_growing, n=149 cells; MyoVI KD_SS, n=150 cells, from three independent experiments. ns, not significant; * P<0,05; **** P<0,0001 by Kruskal-Wallis test.


( C ) IB analysis of OFD1 after serum starvation in control and myosin VI - depleted cells . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI . After four days , cells were serum starved for 24 hours ( SS ) . Lysates were analysed by IB with anti - OFD1 , anti - myosin VI , anti - p62 , and anti - LC3 antibodies . Anti - GAPDH was used as loading control . The amount of OFD1 protein was normalised against GAPDH signal and is expressed as percentage of OFD1 levels in cells grown in serum - starved conditions compared to cells grown in media containing serum . Bars represent mean ± SD . n = 5 independent experiments . * P < 0 , 05 by Kruskal - Wallis test .

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(C) IB analysis of OFD1 after serum starvation in control and myosin VI-depleted cells. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were serum starved for 24 hours (SS). Lysates were analysed by IB with anti-OFD1, anti-myosin VI, anti-p62, and anti-LC3 antibodies. Anti-GAPDH was used as loading control. The amount of OFD1 protein was normalised against GAPDH signal and is expressed as percentage of OFD1 levels in cells grown in serum-starved conditions compared to cells grown in media containing serum. Bars represent mean ±SD. n=5 independent experiments. * P<0,05 by Kruskal-Wallis test.


( D ) IF analysis of primary cilium upon serum starvation . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI . After four days , cells were fixed ( growing ) or serum starved for 24 hours ( SS ) . Cells were immunostained with anti - acetylated tubulin ( to identify the cilia ) , anti - Cep135 ( to identify the centrioles ) and DAPI . Left , representative images , scale bar , 2 μm . Right , results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . n = 3 independent experiments . 100 - 200 cells / condition were counted for each experiment . * * * P < 0 . 001 by two - way ANOVA test .

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(D) IF analysis of primary cilium upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-acetylated tubulin (to identify the cilia), anti-Cep135 (to identify the centrioles) and DAPI. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. n=3 independent experiments. 100-200 cells/condition were counted for each experiment. ***P < 0.001 by two-way ANOVA test.


A Infiltration of GRIp65 - 84 induced cell death similar to GRIp31 - 96 . Bacterially produced 37 nM GST , GST ‐ GRIp31 - 96 or biochemically pure GRI ‐ peptides ( GRIp31 - 96 , GRIp31 - 51 , GRIp47 - 68 , GRIp65 - 84 , GRIp80 - 96 ) were infiltrated into leaves of Col ‐ 0 plants .

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A Infiltration of GRIp65-84 induced cell death similar to GRIp31-96. Bacterially produced 37 nM GST, GST‐GRIp31-96 or biochemically pure GRI‐peptides (GRIp31-96, GRIp31-51, GRIp47-68, GRIp65-84, GRIp80-96) were infiltrated into leaves of Col‐0 plants.


B Infiltration of increasing concentrations of GRIp65 - 84 into Col ‐ 0leaves , electrolyte leakage was measured after 12 h . Background ( red line ) shows ion leakage from infiltration of leaves with increasing concentrations of ( inactive ) GRIp80 - 96 .

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B Infiltration of increasing concentrations of GRIp65-84 into Col‐0leaves, electrolyte leakage was measured after 12 h. Background (red line) shows ion leakage from infiltration of leaves with increasing concentrations of (inactive) GRIp80-96.


C Infiltration of leaves with 37 nM GRIp31 - 96 induced elevated ion leakage in Col ‐ 0 and prk4 , but not in prk5 ‐ 1 or prk5 ‐ 2 . Infiltration with GST caused the same background effect for all lines .

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C Infiltration of leaves with 37 nM GRIp31-96 induced elevated ion leakage in Col‐0 and prk4, but not in prk5‐1 or prk5‐2. Infiltration with GST caused the same background effect for all lines.


D Genomic complementation of prk5 rescues the insensitivity to induction of elevated ion leakage by GRIp31 - 96 .

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D Genomic complementation of prk5 rescues the insensitivity to induction of elevated ion leakage by GRIp31-96.


E Enzymatic superoxide production from xanthine / xanthine oxidase ( XXO ) induced more electrolyte leakage in gri compared to Col ‐ 0 or prk5 ‐ 1 , prk5 ‐ 2 and prk4 after infiltration into leaves . Infiltration with xanthine buffer ( X ) was used as a control .

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E Enzymatic superoxide production from xanthine/xanthine oxidase (XXO) induced more electrolyte leakage in gri compared to Col‐0 or prk5‐1, prk5‐2 and prk4 after infiltration into leaves. Infiltration with xanthine buffer (X) was used as a control.


B PRK5 ‐ CFP localized to the cell periphery in Col ‐ 0mesophyll protoplasts . C - F Co ‐ localization of PRK5 ‐ CFP and PM localized CAAX ‐ YFP in Col ‐ 0mesophyll protoplasts . ( C ) overlay ; ( D ) PRK5 ‐ CFP ( 465 - 510 nm ) ; ( E ) CAAX ‐ YFP ( 521 - 587 nm ) ; ( F ) chloroplast ( 636 - 711 nm ) .

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B PRK5‐CFP localized to the cell periphery in Col‐0mesophyll protoplasts.C-F Co‐localization of PRK5‐CFP and PM localized CAAX‐YFP in Col‐0mesophyll protoplasts. (C) overlay; (D) PRK5‐CFP (465-510 nm); (E) CAAX‐YFP (521-587 nm); (F) chloroplast (636-711 nm).


H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P ‐ ATP and myelin ‐ basic protein ( MBP ) as a substrate in the presence of 10 mM MnCl2 . GST ‐ PRK5 and GST ‐ PRK4 did not show kinase activity . Mutation of conserved residues in kinase subdomains VIb and VII to reconstitute the consensus kinase domain motif restored GST ‐ PRK5H500DA520G kinase activity . GST ‐ CRK7 was used as a positive control . Upper panel shows autoradiograph , and lower panel shows the Coomassie ‐ stained 15 % SDS - polyacrylamide gel .

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H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P‐ATP and myelin‐basic protein (MBP) as a substrate in the presence of 10 mM MnCl2. GST‐PRK5 and GST‐PRK4 did not show kinase activity. Mutation of conserved residues in kinase subdomains VIb and VII to reconstitute the consensus kinase domain motif restored GST‐PRK5H500DA520G kinase activity. GST‐CRK7 was used as a positive control. Upper panel shows autoradiograph, and lower panel shows the Coomassie‐stained 15% SDS-polyacrylamide gel.


A In vitro ‐ produced 35S ‐ labeled ectodomains of PRK5 ( PRK540 - 281 ) or PRK4 ( PRK440 - 279 ) were incubated with bacterially produced GST , GST ‐ GRI or GST ‐ GRI31 - 96 and purified . GST ‐ GRI31 - 96 but not GST directly bound to the ectodomain of PRK5 . Binding of GST ‐ GRI to PRK5 ectodomain and binding of GST ‐ GRI31 - 96 and GST ‐ GRI to the ectodomain of PRK4 were strongly reduced . Upper part : autoradiograph , lower part : Coomassie ‐ stained 12 % SDS - polyacrylamide gel . Asterisks in the Coomassie ‐ stained gel indicate GST , GST ‐ GRI and GST ‐ GRI31 - 96 , respectively . Supplementary Fig S12 shows a Western blot of the GST ‐ tagged proteins .

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A In vitro‐produced 35S‐labeled ectodomains of PRK5 (PRK540-281) or PRK4 (PRK440-279) were incubated with bacterially produced GST, GST‐GRI or GST‐GRI31-96 and purified. GST‐GRI31-96 but not GST directly bound to the ectodomain of PRK5. Binding of GST‐GRI to PRK5 ectodomain and binding of GST‐GRI31-96 and GST‐GRI to the ectodomain of PRK4 were strongly reduced. Upper part: autoradiograph, lower part: Coomassie‐stained 12% SDS-polyacrylamide gel. Asterisks in the Coomassie‐stained gel indicate GST, GST‐GRI and GST‐GRI31-96, respectively. Supplementary Fig S12 shows a Western blot of the GST‐tagged proteins.


B Infiltration of 37 nM GST , GRIp31 - 96 , GRIp65 - 84 or Y ‐ GRIp65 - 84 into Col ‐ 0 or prk5 ‐ 2 leaves . Tyrosine ‐ labeled GRIp65 - 84 still induced cell death in Col ‐ 0 but not in prk5 ‐ 2 plants .

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B Infiltration of 37 nM GST, GRIp31-96, GRIp65-84 or Y‐GRIp65-84 into Col‐0 or prk5‐2 leaves. Tyrosine‐labeled GRIp65-84 still induced cell death in Col‐0 but not in prk5‐2 plants.


C 125I ‐ labeled Y ‐ GRIp65 - 84 ( 0 . 46 nM ) bound specifically to Col ‐ 0membrane fractions ( light gray bars ) , the binding was significantly reduced in prk5 plants . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background levels ( dark gray bars ; all bars show the average of three samples , triangles show individual data points ) .

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C 125I‐labeled Y‐GRIp65-84 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of three samples, triangles show individual data points).


D Immunoprecipitation of PRK5 ‐ c ‐ myc , PRK4 ‐ c ‐ myc or YFP expressed in protoplasts with rabbit polyclonal anti ‐ c ‐ myc antibody , immunoblotted with mouse monoclonal anti ‐ c ‐ myc antibody .

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D Immunoprecipitation of PRK5‐c‐myc, PRK4‐c‐myc or YFP expressed in protoplasts with rabbit polyclonal anti‐c‐myc antibody, immunoblotted with mouse monoclonal anti‐c‐myc antibody.


E Binding of 125I ‐ Y ‐ GRIp65 - 84 ( 0 . 46 nM ) to immunoprecipitates ( using anti ‐ c ‐ myc antibodies ) from prk5 ‐ 2 protoplasts transfected with PRK5 ‐ c ‐ myc , PRK4 ‐ c ‐ myc or YFP , respectively . Binding was competed out with 10 μM unlabeled Y ‐ GRIp65 - 84 . Bars show the average of two samples , and triangles show individual data points . Western blot is shown in panel ( D ) .

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E Binding of 125I‐Y‐GRIp65-84 (0.46 nM) to immunoprecipitates (using anti‐c‐myc antibodies) from prk5‐2 protoplasts transfected with PRK5‐c‐myc, PRK4‐c‐myc or YFP, respectively. Binding was competed out with 10 μM unlabeled Y‐GRIp65-84. Bars show the average of two samples, and triangles show individual data points. Western blot is shown in panel (D).


F Analysis of 125I ‐ Y ‐ GRIp65 - 84 ( 0 . 46 nM ) binding competed out with increasing amounts of unlabeled Y ‐ GRIp65 - 84 to Col ‐ 0membrane extracts . Fifty percent inhibition ( IC50 ) occurred at 25 . 2 nM . Red line shows binding average competition according to a sigmoid curve , dotted lines show 95 % confidence intervals , and circles show data points .

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F Analysis of 125I‐Y‐GRIp65-84 (0.46 nM) binding competed out with increasing amounts of unlabeled Y‐GRIp65-84 to Col‐0membrane extracts. Fifty percent inhibition (IC50) occurred at 25.2 nM. Red line shows binding average competition according to a sigmoid curve, dotted lines show 95% confidence intervals, and circles show data points.


G Excess of GRIp65 - 84 ( 10 μM ) but not of GRIp31 - 96 or other peptides ( GRIp31 - 51 , GRIp47 - 68 , GRIp80 - 96 ) competed the binding of 0 . 46 nM 125I ‐ Y ‐ GRIp65 - 84 to membrane extracts from Col ‐ 0 ( all bars show the average of two samples , triangles show individual data points ) .

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G Excess of GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp65-84 to membrane extracts from Col‐0 (all bars show the average of two samples, triangles show individual data points).


A Recombinant AtMC9 ( rAtMC9 ) cleaved GRI25 - 168in vitro . Bacterially produced MBP ‐ GRI25 - 168 ( from 0 to 2 pmol left to right ) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut ( rAtMC9C147AC29A ) , and cleavage products were analyzed by Western blot with anti ‐ MBP and anti ‐ AtMC9 antibodies . Arrowheads in the anti ‐ MBP blot ( from top to bottom ) indicate MBP ‐ GRI25 - 168 ( 63 . 5 kDa ) , MBPMCS ( 53 . 5 kDa ) , rAtMC9 ‐ cleaved MBP ‐ GRI25 - 168 ( 46 kDa ) . Arrowheads in the anti ‐ AtMC9 blot indicate : 1 : N ‐ terminal domain + p20 + p10 subunits of AtMC9 , 2 : p20 + p10 subunits of AtMC9 , 3 : N ‐ terminal domain + p20 subunit of AtMC9 , 4 : p20 subunit of AtMC9 .

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A Recombinant AtMC9 (rAtMC9) cleaved GRI25-168in vitro. Bacterially produced MBP‐GRI25-168 (from 0 to 2 pmol left to right) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut (rAtMC9C147AC29A), and cleavage products were analyzed by Western blot with anti‐MBP and anti‐AtMC9 antibodies. Arrowheads in the anti‐MBP blot (from top to bottom) indicate MBP‐GRI25-168 (63.5 kDa), MBPMCS (53.5 kDa), rAtMC9‐cleaved MBP‐GRI25-168 (46 kDa). Arrowheads in the anti‐AtMC9 blot indicate: 1: N‐terminal domain + p20 + p10 subunits of AtMC9, 2: p20 + p10 subunits of AtMC9, 3: N‐terminal domain + p20 subunit of AtMC9, 4: p20 subunit of AtMC9.


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B Infiltration of wild‐type, prk5‐1 and atmc9‐1 leaves with 37 nM GRIp31-96, GRIp65-84, GRIp68-78 or GST. GRIp65-84 and GRIp68-78 but not GRIp31-96 were able to induce elevated ion leakage in the atmc9‐1 mutant.


D Infiltration of Col ‐ 0leaves with 37 nM of GRIp65 - 84 , GRIp68 - 78 , Y ‐ GRIp68 - 78 , GRIp31 - 96 or GST . Y ‐ GRIp68 - 78 showed similar activity in cell death induction compared to the other GRI ‐ derived peptides .

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D Infiltration of Col‐0leaves with 37 nM of GRIp65-84, GRIp68-78, Y‐GRIp68-78, GRIp31-96 or GST. Y‐GRIp68-78 showed similar activity in cell death induction compared to the other GRI‐derived peptides.


E 125I ‐ labeled Y ‐ GRIp68 - 78 ( 0 . 46 nM ) bound specifically to Col ‐ 0membrane fractions ( light gray bars ) , the binding was significantly reduced in prk5 ‐ 1 and prk5 ‐ 2 plants . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background levels ( dark gray bars ; all bars show the average of four samples , triangles show individual data points ) .

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E 125I‐labeled Y‐GRIp68-78 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5‐1 and prk5‐2 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of four samples, triangles show individual data points).


F Excess of GRIp68 - 78 and GRIp65 - 84 ( 10 μM ) but not of GRIp31 - 96 or other peptides ( GRIp31 - 51 , GRIp47 - 68 , GRIp80 - 96 ) competed the binding of 0 . 46 nM 125I ‐ Y ‐ GRIp68 - 78 to membrane extracts from Col ‐ 0 ( all bars show average of four samples , triangles show individual data points ) .

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F Excess of GRIp68-78 and GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp68-78 to membrane extracts from Col‐0 (all bars show average of four samples, triangles show individual data points).


G Saturation binding curve for 125I ‐ Y ‐ GRIp68 - 78 to Col ‐ 0membrane extracts . Specific binding was calculated by subtracting non ‐ specific binding from the total binding . The affinity of 125I ‐ Y ‐ GRIp68 - 78 to the receptor ( Kd = 1 . 9 nM ) was calculated by non ‐ linear regression analysis . Scatchard plot is shown in Supplementary Fig SI7 .

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G Saturation binding curve for 125I‐Y‐GRIp68-78 to Col‐0membrane extracts. Specific binding was calculated by subtracting non‐specific binding from the total binding. The affinity of 125I‐Y‐GRIp68-78 to the receptor (Kd = 1.9 nM) was calculated by non‐linear regression analysis. Scatchard plot is shown in Supplementary Fig SI7.


C . Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP - Cwh43 , GFP - Cwh43 - k696fs or GFP - Cwh43 - Leu533ter fusion proteins . To label the ER , HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP - calreticulin fusion protein containing an ER retention signal ( RFP - calreticulin - KDEL ) . To label the Golgi apparatus , cells were immunostained for golgin - 97 ( red ) . When compared with wild type GFP - Cwh43 , GFP - Cwh43 - k696fs and GFP - Cwh43 - Leu533 showed decreased association with the Golgi apparatus ( but not the ER ) and were diffusely distributed throughout the cytoplasm . Scale bar is approximately 5 µm .

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C. Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP-Cwh43, GFP-Cwh43-k696fs or GFP-Cwh43-Leu533ter fusion proteins. To label the ER, HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP-calreticulin fusion protein containing an ER retention signal (RFP-calreticulin-KDEL). To label the Golgi apparatus, cells were immunostained for golgin-97 (red). When compared with wild type GFP-Cwh43, GFP-Cwh43-k696fs and GFP-Cwh43-Leu533 showed decreased association with the Golgi apparatus (but not the ER) and were diffusely distributed throughout the cytoplasm. Scale bar is approximately 5 µm.


D . Western blot analysis of total membrane , aqueous and lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout ( KO ) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced . Cells were transfected to overexpress a control GFP plasmid , a plasmid encoding human wild type Cwh43 with GFP fused to the N - terminus , or a plasmid encoding human CWH43 harboring the iNPH - associated mutation ( Lys696AsnfsTer23 ) with GFP fused to the N - terminus . The Western blot was stained using an antibody directed against CD59 , a GPI - anchored protein .

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D. Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout (KO) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced. Cells were transfected to overexpress a control GFP plasmid, a plasmid encoding human wild type Cwh43 with GFP fused to the N-terminus, or a plasmid encoding human CWH43 harboring the iNPH-associated mutation (Lys696AsnfsTer23) with GFP fused to the N-terminus. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.


Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell . Cilia were visualized using an antibody for acetylated alpha tubulin ( green ) . Cwh43 immunoreactivity was visualized using a specific anti - Cwh43 antibody ( red ) . Scale bar ( left column ) is approximately 4 µm . Images in the column on the right represent a Z - stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell . Scale bar ( right column ) is approximately 5 µm .

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Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell. Cilia were visualized using an antibody for acetylated alpha tubulin (green). Cwh43 immunoreactivity was visualized using a specific anti-Cwh43 antibody (red). Scale bar (left column) is approximately 4 µm. Images in the column on the right represent a Z-stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell. Scale bar (right column) is approximately 5 µm.


A . Scatter plot comparing ventricular volume from CWH43WT / WT ( wild type , WT ) , heterozygous CWH43WT / M533 , homozygous CWH43M533 / M533 and CWH43M533 / A530 mice at 6 months . Ventricular volume was calculated from T2 - weighted MR images of the mouse brain using a custom automated computer algorithm . Horizontal bars indicate the mean of the measurements in each column . Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t - test ( P = 0 . 0015 for CWH43WT / M533 ; P = 0 . 0014 for CWH43M533 / M533 ; P = 0 . 006 for CWH43M533 / A530 ) .

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A. Scatter plot comparing ventricular volume from CWH43WT/WT (wild type, WT), heterozygous CWH43WT/M533, homozygous CWH43M533/M533 and CWH43M533/A530 mice at 6 months. Ventricular volume was calculated from T2-weighted MR images of the mouse brain using a custom automated computer algorithm. Horizontal bars indicate the mean of the measurements in each column. Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t-test (P=0.0015 for CWH43WT/M533; P=0.0014 for CWH43M533/M533; P=0.006 for CWH43M533/A530).


D . Box plot showing rotarod performance data for CWH43WT / WT , CWH43WT / M533 , CWH43M533 / M533 and CWH43M533 / A530 mice at 7 months of age . Data shown are the mean , 1st quartile , 3rd quartile , minimum and maximum for each group of mice . Statistical significance was determined using the unpaired t - test . When compared to wild type CWH43WT / WT mice ( n = 10 ) , balance time on the rotarod was decreased significantly among heterozygous CWH43WT / M533 mice ( * P = 0 . 04 , n = 8 ) , homozygous CWH43M533 / M533 mice ( * P = 0 . 03 , n = 7 ) , and compound heterozygous CWH43M533 / A530 mice ( * P = 0 . 03 , n = 4 ) .

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D. Box plot showing rotarod performance data for CWH43WT/WT, CWH43WT/M533, CWH43M533/M533 and CWH43M533/A530 mice at 7 months of age. Data shown are the mean, 1st quartile, 3rd quartile, minimum and maximum for each group of mice. Statistical significance was determined using the unpaired t-test. When compared to wild type CWH43WT/WT mice (n=10), balance time on the rotarod was decreased significantly among heterozygous CWH43WT/M533 mice (*P=0.04, n=8), homozygous CWH43M533/M533 mice (*P=0.03, n=7), and compound heterozygous CWH43M533/A530 mice (*P=0.03, n=4).


Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533 / M533 mice . Scale bar is approximately 40 µm . The graph on the right quantifies the data from the electron micrographs on the left . Data shown are the mean ± SEM . * = P = 0 . 004 , n = 4 , unpaired t - test .

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Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533/M533 mice. Scale bar is approximately 40 µm. The graph on the right quantifies the data from the electron micrographs on the left. Data shown are the mean ± SEM. * = P=0.004, n = 4, unpaired t-test.


Western blot analysis of total membrane , aqueous and lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type , CWH43M533 / M533 and CWH43WT / M533mouse brain or kidney . The Western blot was stained using an antibody directed against CD59 , a GPI - anchored protein .

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Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type, CWH43M533/M533 and CWH43WT/M533mouse brain or kidney. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.


Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT / WT , CWH43M533 / M533 and CWH43M533 / A530 mice . Arrowheads point to apical surfaces of ependymal and choroid plexus cells . Nuclei are counterstained using DAPI ( blue ) . Scale bar is approximately 5 µm .

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Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. Arrowheads point to apical surfaces of ependymal and choroid plexus cells. Nuclei are counterstained using DAPI (blue). Scale bar is approximately 5 µm.


( B ) Representative images of importin β cargo gradients detected by Rango - 3 FLIM / FRET imaging . Scale bars , 20µm .

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

(B) Representative images of importin β cargo gradients detected by Rango-3 FLIM/FRET imaging. Scale bars, 20µm.


( C ) Line scans of Rango - 3 FRET efficiency ( E ) in 10 µm - wide

(C) Line scans of Rango-3 FRET efficiency (E) in 10 µm-wide areas


( D - F ) Quantification of the Rango - 3 E gradients ( top panels ) and average cellular Rango - 3 E ( bottom ) in involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) protein or treated with 20 µM IPZ . Data for each panel are from at least 2 separate experiments . Means ± SDs , t - test , oocyte numbers indicated in brackets .

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

(D-F) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) in involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20 µM IPZ. Data for each panel are from at least 2 separate experiments. Means ± SDs, t-test, oocyte numbers indicated in brackets.


( G ) Nonlinear single - exponential regression was used to fit the average of radial Rango - 3 τdonor line scans in control oocyte with the one - phase decay model . The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nonlinear single-exponential regression was used to fit the average of radial Rango-3 τdonor line scans in control oocyte with the one-phase decay model. The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell.


( H ) Immunoblotting in biochemical pull - downs with recombinant Ran proteins added to human DL D1 cell lysates . A representative of at least 5 repeats .

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

(H) Immunoblotting in biochemical pull-downs with recombinant Ran proteins added to human DL D1 cell lysates. A representative of at least 5 repeats.


( A ) Quantification of chromosomal cargo gradients ( top ) and average Rango - 3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations . Data from oocytes imaged side - by - side within one experiment . Means ± SDs , one - side ANOVA , Tukey ' s test . Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels .

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

(A) Quantification of chromosomal cargo gradients (top) and average Rango-3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations. Data from oocytes imaged side-by-side within one experiment. Means ± SDs, one-side ANOVA, Tukey's test. Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels.


( C ) Linescans of the estimated Rango - 3 FRET efficiency ( E ) in 10 µm - wide

(C) Linescans of the estimated Rango-3 FRET efficiency (E) in 10 µm-wide areas


( D ) Quantification of the Rango - 3 E gradients ( top panels ) and average cellular Rango - 3 E ( bottom ) oocytes that were treated side - by - side by the injection of mRNA for Rango - 3 , alongside with buffer ( controls ) or 2000ng / µl mRNA for RanT24N or RanT24N , T42A . Means ± SDs , one - side ANOVA , Tukey ' s test , oocyte numbers indicated in brackets .

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

(D) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) oocytes that were treated side-by-side by the injection of mRNA for Rango-3, alongside with buffer (controls) or 2000ng/µl mRNA for RanT24N or RanT24N, T42A. Means ± SDs, one-side ANOVA, Tukey's test, oocyte numbers indicated in brackets.


( B ) Chromosomal gradients detected by RBP3 FLIM / FRET imaging . Scale bars , 20µm .

(B) Chromosomal gradients detected by RBP3 FLIM/FRET imaging. Scale bars, 20µm.


( C ) Line scans of the estimated RBP - 3 in 10 µm - wide

(C) Line scans of the estimated RBP-3 in 10 µm-wide areas


( D - F ) Quantification of the RBP - 3 gradients ( top panels ) and average cellular RBP - 3 E ( bottom ) in separate experiments involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) protein or treated with 20µM IPZ . Means ± SDs , t - test , oocyte numbers indicated in brackets .

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

(D-F) Quantification of the RBP-3 gradients (top panels) and average cellular RBP-3 E (bottom) in separate experiments involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20µM IPZ. Means ± SDs, t-test, oocyte numbers indicated in brackets.


( G ) Nonlinear single - exponential regression was used to fit the average of radial RBP - 3 τdonor line scans in control oocyte with the one - phase decay model .

O O O O O O O O O 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) Nonlinear single-exponential regression was used to fit the average of radial RBP-3 τdonor line scans in control oocyte with the one-phase decay model.


( H ) Images of oocytes expressing GFP - Ran and RCC1 - mCherry from injected mRNAs before FRAP assays . Scale bars , 20 µm .

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(H) Images of oocytes expressing GFP-Ran and RCC1-mCherry from injected mRNAs before FRAP assays. Scale bars, 20 µm.


( I ) RCC1 - mCherry half - time fluorescence recovery ( right , means ± SDs , t - test . ) and an immobile fraction ( left , means ± SDs , Mann - Whitney - test ) in control and IPZ - treated cells , oocyte numbers indicated in brackets .

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(I) RCC1-mCherry half-time fluorescence recovery (right, means ± SDs, t-test.) and an immobile fraction (left, means ± SDs, Mann-Whitney-test) in control and IPZ-treated cells, oocyte numbers indicated in brackets.


( A ) Dose - dependent effects of IPZ on MI spindle assembly are shown by maximum z - projection images from confocal time - lapse imaging of oocytes obtained from transgenic histone H2B - EGFP mice stained with SiR - tubulin . Dashed circles indicate oocyte edges ; 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 B-MEASURED_VAR O O O O O O O O O O O O O

(A) Dose-dependent effects of IPZ on MI spindle assembly are shown by maximum z-projection images from confocal time-lapse imaging of oocytes obtained from transgenic histone H2B-EGFP mice stained with SiR-tubulin. Dashed circles indicate oocyte edges; scale bars, 10 µm.


( B - C ) Live - cell imaging of oocytes was used to quantify the effects of IPZ on GVBD ( B ) and anaphase I entry ( C ) . Anaphase I was defined as the first time point when chromosome segregation was visible . Fisher ' s exact test , oocyte numbers are indicated in brackets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Live-cell imaging of oocytes was used to quantify the effects of IPZ on GVBD (B) and anaphase I entry (C). Anaphase I was defined as the first time point when chromosome segregation was visible. Fisher's exact test, oocyte numbers are indicated in brackets.


( D ) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars .

O O O 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) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars.


( E ) MI spindle formation in oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin matured in the presence of 15 µM IPZ . Representative still images of maximum z - projections are shown . Time in hh : mm is relative to GVBD . Scale bar , 10 µm .

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

(E) MI spindle formation in oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin matured in the presence of 15 µM IPZ. Representative still images of maximum z-projections are shown. Time in hh:mm is relative to GVBD. Scale bar, 10 µm.


( F ) MT intensity at MTOCs at the time of GVBD quantified from SiR - tubulin signal that was normalized to mean cytoplasmic SiR - tubulin signal . Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group ( means ± SDs , Mann - Whitney - test ) . Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ - treated oocytes were analyzed . ( G ) Time course of the MT intensity in the area of spindle after GVBD . Mean of MT intensity was arbitrarily centered to 1 in the control group ( means ± SDs ) . Oocyte numbers are indicated in brackets .

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

(F) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group (means ± SDs, Mann-Whitney-test). Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ-treated oocytes were analyzed. (G) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group (means ± SDs). Oocyte numbers are indicated in brackets.


( H ) Oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin were treated with 15 µM IPZ after being cultured for 5h in control media . Maximum intensity z - projections from confocal live - cell imaging are shown . Time in hh : mm is relative to GVBD , DMSO or 15 uM IPZ was added at time 5 : 00 . Scale bars , 10 µm .

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

(H) Oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin were treated with 15 µM IPZ after being cultured for 5h in control media. Maximum intensity z-projections from confocal live-cell imaging are shown. Time in hh:mm is relative to GVBD, DMSO or 15 uM IPZ was added at time 5:00. Scale bars, 10 µm.


( I ) . Spindle volume at 1h and metaphase I after IPZ addition . The metaphase time was defined as the last time point before anaphase or , in the IPZ - treated oocytes that failed anaphase entry , as the average control metaphase time . Means ± SDs , Mann - Whitney test , oocyte numbers are indicated in brackets .

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

(I). Spindle volume at 1h and metaphase I after IPZ addition. The metaphase time was defined as the last time point before anaphase or, in the IPZ-treated oocytes that failed anaphase entry, as the average control metaphase time. Means ± SDs, Mann-Whitney test, oocyte numbers are indicated in brackets.


( J ) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media . Oocyte numbers are indicated in brackets .

(J) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media. Oocyte numbers are indicated in brackets.


( A ) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B - mCherry from microinjected mRNA , co - stained with SiR - tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2 . Maximum intensity z - stack projections from confocal live - cell movies 30 min after GVBD , at metaphase I and anaphase I . Time in hh : mm is relative to GVBD . 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-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B-mCherry from microinjected mRNA, co-stained with SiR-tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2. Maximum intensity z-stack projections from confocal live-cell movies 30 min after GVBD, at metaphase I and anaphase I. Time in hh: mm is relative to GVBD. Scale bars, 10 µm.


( B - C ) The frequency of GVBD ( B ) and anaphase I entry ( C ) quantified by live confocal imaging of oocytes stained and treated Fisher ' s exact test , oocyte numbers are shown in brackets . ( D ) Changes in spindle volume over time after GVBD . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) The frequency of GVBD (B) and anaphase I entry (C) quantified by live confocal imaging of oocytes stained and treated Fisher's exact test, oocyte numbers are shown in brackets. (D) Changes in spindle volume over time after GVBD. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.


( A ) The effects of RanT24N , T42A , and RanT24N on MI spindle assembly observed in maximum z - projection images from confocal time - lapse imaging of oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin . Time in hh : mm is relative to GVBD . Scale bar , 10 µm . ( B , C ) Live - cell imaging of oocytes as in ( A ) was used to quantify the effects of Ran mutant . Means ± SDs . , Mann - Whitney test , oocyte numbers are indicated in brackets .

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

(A) The effects of RanT24N, T42A, and RanT24N on MI spindle assembly observed in maximum z-projection images from confocal time-lapse imaging of oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin. Time in hh:mm is relative to GVBD. Scale bar, 10 µm. (B, C) Live-cell imaging of oocytes as in (A) was used to quantify the effects of Ran mutant. Means ± SDs., Mann-Whitney test, oocyte numbers are indicated in brackets.


( D ) MT intensity at MTOCs at the time of GVBD quantified from SiR - tubulin signal that was normalized to mean cytoplasmic SiR - tubulin signal . Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group . Means ± SDs , Kruskal - Wallis , and Dunn ' s tests . 25 control oocytes with 264 MTOCs in total , 10 RanT24N - expression oocytes with 70 MTOCs in total and 19 RanT24N , T42A - expressing oocytes with 96 MTOCs in total were analyzed .

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

(D) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group. Means ± SDs, Kruskal-Wallis, and Dunn's tests. 25 control oocytes with 264 MTOCs in total, 10 RanT24N-expression oocytes with 70 MTOCs in total and 19 RanT24N, T42A-expressing oocytes with 96 MTOCs in total were analyzed.


( E ) Time course of the MT intensity in the area of spindle after GVBD . Mean of MT intensity was arbitrarily centered to 1 in the control group . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets .

O O O O O O O O O O O O O O O O O O O O O O O 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) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.


( F ) Frequency and severity of MI spindle phenotypes in the oocytes expressing RanT24N or RanT24N , T42A . Oocyte numbers are shown in brackets .

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(F) Frequency and severity of MI spindle phenotypes in the oocytes expressing RanT24N or RanT24N, T42A. Oocyte numbers are shown in brackets.


( G , H ) Quantification of the percentage of oocytes ( G ) with chromosome segregation errors in anaphase I and ( H ) with defective MTOC coalescence during metaphase I . Means ± SDs , oocyte numbers are shown in brackets .

O O O O O O O O O O O O O O O O O O O O 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, H) Quantification of the percentage of oocytes (G) with chromosome segregation errors in anaphase I and (H) with defective MTOC coalescence during metaphase I. Means ± SDs, oocyte numbers are shown in brackets.


( I ) Detection of MTs and pericentrin - containing MTOCs by IF in control and RanT24N , T42A - expressing oocytes . Arrow indicates unaligned chromosome . ( J ) Quantification of chromosomal spread area in oocytes depicted in ( I ) , Means ± SDs , Mann - Whitney test , oocyte numbers indicated in brackets .

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(I) Detection of MTs and pericentrin-containing MTOCs by IF in control and RanT24N, T42A - expressing oocytes. Arrow indicates unaligned chromosome. (J) Quantification of chromosomal spread area in oocytes depicted in (I), Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.


( A ) . Maximum intensity z - stack projections of confocal IF images ( DNA stain , indirect IF for tubulin and TPX2 ) in untreated oocytes ( control ) , oocytes expressing RanT24N or injected with importin β ( 71 - 876 ) . The representative of at least 5 experiments is shown .

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

(A). Maximum intensity z-stack projections of confocal IF images (DNA stain, indirect IF for tubulin and TPX2) in untreated oocytes (control), oocytes expressing RanT24N or injected with importin β(71-876). The representative of at least 5 experiments is shown.


( B - D ) Maximum intensity z - stack projections of IF images in control and importin β ( 71 - 876 ) - injected cells were used to quantify the spindle vs . whole - cell area fraction ( B ) and the ratios of the average signal in the spindle vs . cytoplasm for the tubulin ( C ) and TPX2 ( D ) staining . Means ± SDs , with t - test in ( B , C ) and Mann - Whitney test in ( D ) , oocyte numbers indicated in brackets .

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

(B-D) Maximum intensity z-stack projections of IF images in control and importin β(71-876)-injected cells were used to quantify the spindle vs. whole-cell area fraction (B) and the ratios of the average signal in the spindle vs. cytoplasm for the tubulin (C) and TPX2 (D) staining. Means ± SDs, with t-test in (B, C) and Mann-Whitney test in (D), oocyte numbers indicated in brackets.


( E ) Ratios of the spindle - associated fractions of TPX2 vs . tubulin were calculated in maximum intensity z - stack projections of IF images . Means ± SDs , Mann - Whitney test , oocyte numbers indicated in brackets .

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

(E) Ratios of the spindle-associated fractions of TPX2 vs. tubulin were calculated in maximum intensity z-stack projections of IF images. Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.


( F ) Maximum intensity z - stack projections of IF images were used to quantify the number of ectopic MT asters . Means ± SDs , ANOVA with Kruskal - Wallis and Dunn ' s tests , oocyte numbers indicated in brackets .

(F) Maximum intensity z-stack projections of IF images were used to quantify the number of ectopic MT asters. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests, oocyte numbers indicated in brackets.


( G ) Maximum intensity z - stack projections of confocal IF images stained for DNA , pericentrin , and tubulin ( directly labeled primary antibody ) . A representative example of at least 2 experiments is shown . Scale bar , 20 µm . ( H - I ) The number and size of pericentrin foci were quantified in maximum - intensity z - stack projections of IF images stained as in ( G ) in at least 2 separate experiments . Means ± SDs , ANOVA with Kruskal - Wallis and Dunn ' s tests .

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

(G) Maximum intensity z-stack projections of confocal IF images stained for DNA, pericentrin, and tubulin (directly labeled primary antibody). A representative example of at least 2 experiments is shown. Scale bar, 20 µm. (H-I) The number and size of pericentrin foci were quantified in maximum-intensity z-stack projections of IF images stained as in (G) in at least 2 separate experiments. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests.


( A ) 22 Tie1 - binding antibodies were screened for phospho - AKT levels in an in vitro Ang1 - stimulation experiment . Only one clone ( AB - Tie1 - 39 ) reduced AKT phosphorylation indicating suppressed Tie2 signaling ( n = 1 experiment ) .

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

(A) 22 Tie1-binding antibodies were screened for phospho-AKT levels in an in vitro Ang1-stimulation experiment. Only one clone (AB-Tie1-39) reduced AKT phosphorylation indicating suppressed Tie2 signaling (n = 1 experiment).


( C ) Tumor growth curves show delayed primary tumor growth upon treatment with AB - Tie1 - 39 antibody as compared to IgG treatment ( mean ± SEM , n = 9 mice ) . * , P < 0 . 05 ; * * * , P < 0 . 001 ( 2 - way ANNOVA test ) .

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(C) Tumor growth curves show delayed primary tumor growth upon treatment with AB-Tie1-39 antibody as compared to IgG treatment (mean ± SEM, n = 9 mice). *, P<0.05; ***, P<0.001 (2-way ANNOVA test).


( D ) Representative immunofluorescence images of tumor sections stained with CD31 ( EC - specific marker ) and Desmin ( mural cell - specific marker ) . Scale bars = 200 μm .

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

(D) Representative immunofluorescence images of tumor sections stained with CD31 (EC-specific marker) and Desmin (mural cell-specific marker). Scale bars = 200 μm.


( E ) Dot plots show quantitation of intratumoral vessel density , mural cell coverage using Desmin and aSMA co - staining , and tumor necrosis ( mean ± SD , n = 9 mice ) . All comparisons were rendered non - significant according to two - tailed Mann - Whitney U test .

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(E) Dot plots show quantitation of intratumoral vessel density, mural cell coverage using Desmin and aSMA co-staining, and tumor necrosis (mean ± SD, n = 9 mice). All comparisons were rendered non-significant according to two-tailed Mann-Whitney U test.


( A ) Following a neoadjuvant treatment regimen with IgG or AB - Tie1 - 39 , mice were monitored by whole - body bioluminescence imaging . Shown are the images acquired two weeks after primary tumor resection .

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(A) Following a neoadjuvant treatment regimen with IgG or AB-Tie1-39, mice were monitored by whole-body bioluminescence imaging. Shown are the images acquired two weeks after primary tumor resection.


( B ) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging ( mean ± SD , nIgG = 8 , nAB - Tie1 - 39 = 9 mice ) . * , P < 0 . 05 ; * * , P < 0 . 01 ( two - tailed Mann - Whitney U test ) .

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(B) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging (mean ± SD, nIgG = 8, nAB-Tie1-39 = 9 mice). *, P<0.05; **, P<0.01 (two-tailed Mann-Whitney U test).


( D ) Kaplan - Meier graph comparing percent survival between mice treated with either IgG or AB - Tie1 - 39 ( nIgG = 10 , nAB - Tie1 - 39 = 9 mice ) . * * , P < 0 . 01 ( Log - rank ( Mantel - Cox ) test ) .

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(D) Kaplan-Meier graph comparing percent survival between mice treated with either IgG or AB-Tie1-39 (nIgG = 10, nAB-Tie1-39 = 9 mice). **, P<0.01 (Log-rank (Mantel-Cox) test).


( B ) Representative images of the whole lungs are shown on the left . Scale bars = 5 mm . Quantitation of the number of lung metastatic foci is plotted on the right ( mean ± SD , nIgG = 9 , nAB - Tie1 - 39 = 8 mice ) . * , P < 0 . 05 ( two - tailed Mann - Whitney U test ) .

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(B) Representative images of the whole lungs are shown on the left. Scale bars = 5 mm. Quantitation of the number of lung metastatic foci is plotted on the right (mean ± SD, nIgG = 9, nAB-Tie1-39 = 8 mice). *, P<0.05 (two-tailed Mann-Whitney U test).


( C ) EC were seeded on gelatin - coated Transwell inserts . RFP - labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer . Thereafter , transmigrated tumor cells were counted . The dot plot presents the normalized data ( mean ± SD , n = 9 inserts from three independent experiments ) . * * * , P < 0 . 001 ( two - tailed Mann - Whitney U test ) .

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(C) EC were seeded on gelatin-coated Transwell inserts. RFP-labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***, P<0.001 (two-tailed Mann-Whitney U test).


( D ) LLC tumor - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung tissue lysates were immunoblotted with anti - phospho - TIE2 , total - TIE2 and ACTB ( upper panel ; n = 6 mice ) . Densitometric quantitation of the blots presented in the upper panel is shown ( mean ± SD , n = 6 mice ) . * * , P < 0 . 01 ( two - tailed Mann - Whitney U test ) .

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(D) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung tissue lysates were immunoblotted with anti-phospho-TIE2, total-TIE2 and ACTB (upper panel; n = 6 mice). Densitometric quantitation of the blots presented in the upper panel is shown (mean ± SD, n = 6 mice). **, P<0.01 (two-tailed Mann-Whitney U test).


( E ) LLC tumor - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung EC were FACS - sorted and used for global gene expression ( n = 4 mice ) . On the left , a heatmap shows significantly - regulated genes ( red outline = AB - Tie1 - 39 - treated ; black outline = IgG - treated ) . On the right , top significantly regulated bio - functions in AB - Tie1 - 39 - treated as compared to IgG - treated mice in Ingenuity Pathway Analysis .

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(E) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung EC were FACS-sorted and used for global gene expression (n = 4 mice). On the left, a heatmap shows significantly- regulated genes (red outline = AB-Tie1-39-treated; black outline = IgG-treated). On the right, top significantly regulated bio-functions in AB-Tie1-39-treated as compared to IgG-treated mice in Ingenuity Pathway Analysis.


A ) Murine small intestines immunostained for the proliferation marker Ki67 , apoptosis marker cleaved caspase 3 ( Cas3 ) , or YAP from 4 control ( Yapfl / fl Tazfl / fl ) and 4 YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a moderate increase in apoptosis ( Cas3 + ) in crypt base stem cells , quantified on the right , but no overall effect on the morphology of the small intestine . Arrows point to apoptotic cells . B ) Murine large intestines immunostained for proliferation marker Ki67 , apoptosis marker cleaved caspase 3 ( Cas3 ) , or YAP from control ( Yapfl / fl Tazfl / fl ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to an increased number of apoptotic ( Cas3 + ) cells within the transverse folds of the ascending colon .

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A) Murine small intestines immunostained for the proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from 4 control (Yapfl/fl Tazfl/fl) and 4 YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a moderate increase in apoptosis (Cas3+) in crypt base stem cells, quantified on the right, but no overall effect on the morphology of the small intestine. Arrows point to apoptotic cells. B) Murine large intestines immunostained for proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from control (Yapfl/fl Tazfl/fl) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to an increased number of apoptotic (Cas3+) cells within the transverse folds of the ascending colon.


C ) Murine small intestines immunostained for YAP or cleaved caspase 3 ( Cas3 ) from control ( Villin - CreERt ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a mild increase in apoptosis ( Cas3 + ) in crypt base stem cells , but no overall effect on the morphology of the small intestine . Arrows point to stem cells ( YAP nuclear staining ) , while arrows point to apoptotic cells ( Cas3 staining ) . N > 10 animals per genotype . D ) Murine large intestines immunostained for YAP or cleaved caspase 3 ( Cas3 ) from control ( Villin - CreERt ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to increased apopotosis ( Cas3 + ) and severe morphological defects in the transverse folds of the ascending colon , but does not affect morphology of the remaining colon regions . Quantitatively , 75 % of n = 4 animals showed this severe phenotype .

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C) Murine small intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a mild increase in apoptosis (Cas3+) in crypt base stem cells, but no overall effect on the morphology of the small intestine. Arrows point to stem cells (YAP nuclear staining), while arrows point to apoptotic cells (Cas3 staining). N > 10 animals per genotype. D) Murine large intestines immunostained for YAP or cleaved caspase 3 (Cas3 ) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to increased apopotosis (Cas3+) and severe morphological defects in the transverse folds of the ascending colon, but does not affect morphology of the remaining colon regions. Quantitatively, 75% of n = 4 animals showed this severe phenotype.


A ) Murine small ( top ) and large ( bottom ) intestines isolated from control ( Cre negative ) Lats1flox / flox Lats2flox / flox animals and Villin - CreERt Lats1flox / flox Lats2flox / flox animals treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt - villus axis in controls , with nuclear YAP and Ki67 positive cells restricted to the crypt base ( representative images from n = 5 mice ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells . Note the gradient of YAP expression levels is maintained along the crypt - villus axis ( representative images from n = 5 mice for each genotype ) . ( Right ) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base . ( Bottom right ) Q - PCR analysis of Wnt pathway target genes reveals that Lats1 / 2 dKO causes a mild reduction in Lgr5 expression , with complete loss of Olfm4 .

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A) Murine small (top) and large (bottom) intestines isolated from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt-villus axis in controls, with nuclear YAP and Ki67 positive cells restricted to the crypt base (representative images from n=5 mice). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells. Note the gradient of YAP expression levels is maintained along the crypt-villus axis (representative images from n=5 mice for each genotype). (Right) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base. (Bottom right) Q-PCR analysis of Wnt pathway target genes reveals that Lats1/2 dKO causes a mild reduction in Lgr5 expression, with complete loss of Olfm4.


B ) Axin2 mRNA expression was measured by RNAscope ( red ) and found to be increased in both intensity and uniformity of staining along the crypt - villus axis in Lats1 / 2 dKO after 3 days post i . p . injection ( dpi ) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2 . At 7dpi , Axin2 mRNA levels remain uniform along the crypt - villus axis in Lats1 / 2 dKO animals , although their total level has declined compared to 3dpi .

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B) Axin2 mRNA expression was measured by RNAscope (red) and found to be increased in both intensity and uniformity of staining along the crypt-villus axis in Lats1/2 dKO after 3 days post i.p. injection (dpi) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2. At 7dpi, Axin2 mRNA levels remain uniform along the crypt-villus axis in Lats1/2 dKO animals, although their total level has declined compared to 3dpi.


D ) Q - PCR analysis of YAP - TEAD and their target genes Ctgf and Cyr61 reveals that Lats1 / 2 dKO causes a strong induction of Ctgf and Cyr61 expression , as expected , as well as a strong induction of the common Wnt and YAP target gene CyclinD1 .

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D) Q-PCR analysis of YAP-TEAD and their target genes Ctgf and Cyr61 reveals that Lats1/2 dKO causes a strong induction of Ctgf and Cyr61 expression, as expected, as well as a strong induction of the common Wnt and YAP target gene CyclinD1.


B ) RNAscope in situ hybridisation analysis of TEAD1 / 2 / 4 mRNA expression from control ( Cre negative ) Lats1flox / flox Lats2flox / flox animals and Villin - CreERt Lats1flox / flox Lats2flox / flox animals treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine ( top ) and colon ( bottom ) . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . ( n = 5 animals for each genotype )

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B) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype)


C ) Q - PCR analysis reveals a progressive increase in the YAP - TEAD target gene Ctgf in Lats1 / 2 dKO intestines at 3 - days and 7 - days post i . p . injection with tamoxifen , as well as similarly increased TEAD1 and TEAD4 expression , confirming that TEAD1 and TEAD4 are YAP - responsive genes . Notably , Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1 / 2 deletion , consistent with Axin2 being a known Wnt - specific target gene , and with Wnt signalling remaining active in Lats1 / 2 dKO intestines .

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C) Q-PCR analysis reveals a progressive increase in the YAP-TEAD target gene Ctgf in Lats1/2 dKO intestines at 3-days and 7-days post i.p. injection with tamoxifen, as well as similarly increased TEAD1 and TEAD4 expression, confirming that TEAD1 and TEAD4 are YAP-responsive genes. Notably, Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1/2 deletion, consistent with Axin2 being a known Wnt-specific target gene, and with Wnt signalling remaining active in Lats1/2 dKO intestines.


A ) RNAscope in situ hybridisation analysis of TEAD1 / 2 / 4 mRNA expression from control ( wild - type ) and ApcMin mutant tumours from the small intestine ( top ) and colon ( bottom ) . All three genes were strongly induced within the ApcMin mutant tumours , with TEAD2 being most strongly induced in colon ApcMin tumours ( n = 3 animals with multiple tumours within the small and large intestines ) .

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A) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (wild-type) and ApcMin mutant tumours from the small intestine (top) and colon (bottom). All three genes were strongly induced within the ApcMin mutant tumours, with TEAD2 being most strongly induced in colon ApcMin tumours (n=3 animals with multiple tumours within the small and large intestines).


C ) Antibody staining for TEAD4 reveals a gradient of expression along the crypt - villus axis in both the small and large intestine , with uniformly high expression in colorectal adenomas , similar to SOX9 .

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C) Antibody staining for TEAD4 reveals a gradient of expression along the crypt-villus axis in both the small and large intestine, with uniformly high expression in colorectal adenomas, similar to SOX9.


B ) YAP immunostaining of 3 - day old wild type organoids treated with DMSO ( Control ) or Porcupine inhibitor LGK974 at 5µM . Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation . Quantification of YAP protein levels is shown below , with statistical significance in an unpaired 2 - tailed t - test .

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B) YAP immunostaining of 3-day old wild type organoids treated with DMSO (Control) or Porcupine inhibitor LGK974 at 5µM. Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation. Quantification of YAP protein levels is shown below, with statistical significance in an unpaired 2-tailed t-test.


C ) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level . Scale bar is 250µm . n > 10 organoids in each experiment . Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right , with statistical significance in an unpaired 2 - tailed t - test .

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C) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level. Scale bar is 250µm. n > 10 organoids in each experiment. Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right, with statistical significance in an unpaired 2-tailed t-test.