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

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

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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 of OFD1 localisation at centrioles . hTERT - RPE1 were transfected with siRNA against myosin VI , immunostained with anti - OFD1 anti - acetylated tubulin antibodies visualised using structured illumination microscopy ( SIM ) . Representative are shown , scale bar , 500 nm . A scheme of estimated localisation of OFD1 in two conditions is depicted on 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 of distal appendage markers FBF1 ODF2 in control myosin VI - depleted . To emphasise symmetry of structures , signals from nine appendages were averaged ( bottom ) . Representative 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 of centriole - associated GFP - OFD1 . hTERT - RPE stably expressing GFP - OFD1 centrin1 - dTomato were transfected with siRNA against myosin VI . After four days , were treated with nocodazole ( 1 hour , 6 μg / ml ) subjected to live - imaging . ( E ) A representative graph of one out of three experiments . For each time point , fraction of recovery of GFP - OFD1 is shown . Results are expressed as means with 95 % confidence interval . n = 12 ( Mock ) , n = 13 ( MyoVI KD ) . ( F ) of half - time of recovery ( t1 / 2 , ) of mobile fraction of GFP - OFD1 . Results are expressed as mean ± SD . n = 41 ( Mock ) , n = 31 ( MyoVI KD ) , from three independent experiments . ns , not significant ; * * * P < 0 , 0005 by Unpaired T - .

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

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

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

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

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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.


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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 ) 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 prk4 , but not in prk5 ‐ 1 or prk5 ‐ 2 . Infiltration with GST caused same background effect for 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 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 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 periphery in Col ‐ 0mesophyll protoplasts . C - F Co ‐ localization of PRK5 ‐ CFP 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 myelin ‐ basic ( MBP ) as a substrate in presence of 10 mM MnCl2 . GST ‐ PRK5 GST ‐ PRK4 did not show kinase activity . Mutation of conserved residues in kinase subdomains VIb VII to reconstitute consensus kinase domain motif restored GST ‐ PRK5H500DA520G kinase activity . GST ‐ CRK7 was used as a positive control . Upper panel shows autoradiograph , lower panel shows 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 purified . GST ‐ GRI31 - 96 but not GST directly bound to ectodomain of PRK5 . Binding of GST ‐ GRI to PRK5 ectodomain binding of GST ‐ GRI31 - 96 GST ‐ GRI to ectodomain of PRK4 were strongly reduced . Upper part : autoradiograph , lower part : Coomassie ‐ stained 12 % SDS - polyacrylamide gel . Asterisks in Coomassie ‐ stained gel indicate GST , GST ‐ GRI GST ‐ GRI31 - 96 , respectively . Supplementary Fig S12 shows a Western blot of GST ‐ tagged .

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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 in Col ‐ 0 but not in prk5 ‐ 2 .

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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 ( gray bars ) , binding was significantly reduced in prk5 . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background ( gray bars ; bars show average of three samples , triangles show 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 average of two samples , triangles show 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 of 125I ‐ Y ‐ GRIp65 - 84 ( 0 . 46 nM ) binding competed out with increasing amounts of unlabeled Y ‐ GRIp65 - 84 to Col ‐ 0membrane extracts . Fifty inhibition ( IC50 ) occurred at 25 . 2 nM . Red shows binding average competition according to a sigmoid curve , dotted lines show 95 % confidence intervals , 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 binding of 0 . 46 nM 125I ‐ Y ‐ GRIp65 - 84 to extracts from Col ‐ 0 ( bars show average of two samples , triangles show 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 ) 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 ) , cleavage products were analyzed by Western blot with anti ‐ MBP anti ‐ AtMC9 antibodies . Arrowheads in anti ‐ MBP blot ( from top to bottom ) indicate MBP ‐ GRI25 - 168 ( 63 . 5 kDa ) , MBPMCS ( 53 . 5 kDa ) , rAtMC9 ‐ MBP ‐ GRI25 - 168 ( 46 kDa ) . Arrowheads in 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 induction compared to 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 ( gray bars ) , binding was significantly reduced in prk5 ‐ 1 prk5 ‐ 2 . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background ( gray bars ; bars show average of four samples , triangles show 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 GRIp65 - 84 ( 10 μM ) but not of GRIp31 - 96 or other peptides ( GRIp31 - 51 , GRIp47 - 68 , GRIp80 - 96 ) competed binding of 0 . 46 nM 125I ‐ Y ‐ GRIp68 - 78 to extracts from Col ‐ 0 ( bars show average of four samples , triangles show 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 total binding . affinity of 125I ‐ Y ‐ GRIp68 - 78 to receptor ( Kd = 1 . 9 nM ) was calculated by non ‐ linear regression . 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 . of HeLa transfected with plasmids encoding human GFP - Cwh43 , GFP - Cwh43 - k696fs or GFP - Cwh43 - Leu533ter fusion . To label ER , HeLa were transduced with a baculovirus encoding a fluorescently labeled RFP - calreticulin fusion containing an ER retention signal ( RFP - calreticulin - KDEL ) . To label Golgi apparatus , were immunostained for golgin - 97 ( red ) . When compared with wild type GFP - Cwh43 , GFP - Cwh43 - k696fs GFP - Cwh43 - Leu533 showed decreased association with Golgi apparatus ( but not ER ) were diffusely distributed throughout 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 of total , aqueous lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type HeLa two independent CRISPR CWH43 knockout ( KO ) HeLa lines in which a mutated CWH43 gene encodes a that is truncated near Leu533 CWH43 mRNA are markedly reduced . were transfected to overexpress a control GFP plasmid , a plasmid encoding human wild type Cwh43 with GFP fused to N - terminus , or a plasmid encoding human CWH43 harboring iNPH - associated mutation ( Lys696AsnfsTer23 ) with GFP fused to N - terminus . Western blot was stained using an antibody directed against CD59 , a GPI - anchored .

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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 immunocytochemistry of a single cultured mouse ciliated ependymal . 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 . in column on right represent a Z - stack reconstruction of confocal showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal . 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 CWH43M533 / A530 mice at 6 months . Ventricular volume was calculated from T2 - weighted MR of mouse brain using a custom automated computer algorithm . Horizontal bars indicate mean of measurements in each column . Statistical significance for each mutant mouse compared to WT was determined using unpaired t - ( 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 CWH43M533 / A530 mice at 7 months of . Data shown are mean , 1st quartile , 3rd quartile , minimum maximum for each group of mice . Statistical significance was determined using unpaired t - . When compared to wild type CWH43WT / WT mice ( n = 10 ) , balance time on rotarod was decreased significantly among heterozygous CWH43WT / M533 mice ( * P = 0 . 04 , n = 8 ) , homozygous CWH43M533 / M533 mice ( * P = 0 . 03 , n = 7 ) , 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 of ependymal surface of lateral ventricle of CWH43 WT CWH43M533 / M533 mice . Scale bar is approximately 40 µm . graph on right quantifies data from electron on left . Data shown are mean ± . * = P = 0 . 004 , n = 4 , unpaired t - .

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

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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.


immunohistochemistry for CD59 in ependymal layer choroid plexus of lateral ventricle from CWH43WT / WT , CWH43M533 / M533 CWH43M533 / A530 mice . Arrowheads point to apical surfaces of ependymal choroid plexus . 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 of importin β cargo gradients detected by Rango - 3 FLIM / FRET imaging . Scale bars , 20µm .

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


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

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(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 average of radial Rango - 3 τdonor scans in control oocyte with one - phase decay model . dashed corresponds to average of spindle pole from chromosomes in same .

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(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 added to human DL D1 lysates . A representative of at least 5 repeats .

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(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 ) of chromosomal cargo gradients ( top ) 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 . Numbers of analyzed oocytes are indicated in brackets in top panel are identical for both panels .

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(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 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 ) of Rango - 3 E gradients ( top panels ) average Rango - 3 E ( bottom ) oocytes that were treated side - by - side by 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 , oocyte numbers indicated in brackets .

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

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


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

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(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 average of radial RBP - 3 τdonor scans in control oocyte with one - phase decay model .

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(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 ) of oocytes expressing GFP - Ran 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 recovery ( right , means ± SDs , t - . ) an immobile fraction ( left , means ± SDs , Mann - Whitney - ) in control IPZ - treated , 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 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 .

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

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(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 severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at top of bars .

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(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 stained with SiR - tubulin matured in presence of 15 µM IPZ . Representative still of maximum z - projections are shown . Time in hh : mm is relative to GVBD . Scale bar , 10 µm .

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

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-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

(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 stained with SiR - tubulin were treated with 15 µM IPZ after being cultured for 5h in control media . Maximum z - projections from confocal live - 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 .

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(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 metaphase I after IPZ addition . metaphase time was defined as last time point before anaphase or , in IPZ - treated oocytes that failed anaphase entry , as average control metaphase time . Means ± SDs , Mann - Whitney , 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

(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 .

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(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 ) rescue of 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 noninjected or injected with mRNAs coding for RanQ69L or hTPX2 . Maximum z - stack projections from confocal live - movies 30 min after GVBD , at metaphase I 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 B-MEASURED_VAR 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

(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 ) frequency of GVBD ( B ) anaphase I entry ( C ) quantified by live confocal imaging of oocytes stained treated Fisher ' s exact , 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 .

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

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

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(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 MT in of spindle after GVBD . Mean of MT was arbitrarily centered to 1 in 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

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

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

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

(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 z - stack projections of confocal IF ( stain , indirect IF for tubulin TPX2 ) in untreated oocytes ( control ) , oocytes expressing RanT24N or injected with importin β ( 71 - 876 ) . 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 B-MEASURED_VAR I-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

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

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

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

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

(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 z - stack projections of IF were used to quantify number of ectopic MT asters . Means ± SDs , ANOVA with Kruskal - Wallis Dunn ' s , 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

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

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

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

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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 of sections stained with CD31 ( EC - specific marker ) Desmin ( mural - specific marker ) . Scale bars = 200 μm .

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(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 coverage using Desmin aSMA co - , necrosis ( mean ± SD , n = 9 mice ) . comparisons were rendered non - significant according to two - Mann - Whitney U .

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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 - bioluminescence imaging . Shown are acquired two after 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 as well as ex vivo lung lymph node bioluminescence imaging ( mean ± SD , nIgG = 8 , nAB - Tie1 - 39 = 9 mice ) . * , P < 0 . 05 ; * * , P < 0 . 01 ( two - Mann - Whitney U ) .

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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 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 ) ) .

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

(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 were allowed to transmigrate for 8 h through endothelial layer . Thereafter , transmigrated were counted . dot plot presents normalized data ( mean ± SD , n = 9 inserts from three independent experiments ) . * * * , P < 0 . 001 ( two - Mann - Whitney U ) .

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

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

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

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 O O O O O O O O O O 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 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 B-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

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

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

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 caspase 3 ( Cas3 ) from control ( Villin - CreERt ) YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) treated with tamoxifen . Tissues were harvested analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a mild increase in apoptosis ( Cas3 + ) in crypt base , but no overall effect on of small intestine . Arrows point to ( YAP ) , while arrows point to apoptotic ( Cas3 ) . N > 10 per genotype . D ) Murine large intestines immunostained for YAP or caspase 3 ( Cas3 ) from control ( Villin - CreERt ) YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) treated with tamoxifen . Tissues were harvested analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to increased apopotosis ( Cas3 + ) severe morphological defects in transverse folds of ascending colon , but does not affect of remaining colon regions . Quantitatively , 75 % of n = 4 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 ) large ( bottom ) intestines isolated from control ( Cre negative ) Lats1flox / flox Lats2flox / flox Villin - CreERt Lats1flox / flox Lats2flox / flox treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Immunostaining for YAP Ki67 shows a gradient of YAP expression along crypt - villus axis in controls , with YAP Ki67 positive restricted to crypt base ( representative 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 YAP immunostaining in epithelial an expanded proliferative zone marked by Ki67 positive . Note gradient of YAP expression is maintained along crypt - villus axis ( representative from n = 5 mice for each genotype ) . ( Right ) Immunostaining for Paneth marker Lyz reveals loss of this marker from crypt base . ( Bottom right ) Q - PCR 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 ) found to be increased in both uniformity of along crypt - villus axis in Lats1 / 2 dKO after 3 days post i . p . injection ( dpi ) with tamoxifen to induce homozygous deletion of Lats1 Lats2 . At 7dpi , Axin2 mRNA remain uniform along crypt - villus axis in Lats1 / 2 dKO , 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 of YAP - TEAD their target genes Ctgf Cyr61 reveals that Lats1 / 2 dKO causes a strong induction of Ctgf Cyr61 expression , as expected , as well as a strong induction of common Wnt 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 of TEAD1 / 2 / 4 mRNA expression from control ( Cre negative ) Lats1flox / flox Lats2flox / flox Villin - CreERt Lats1flox / flox Lats2flox / flox 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 TEAD4 in both small intestine ( top ) colon ( bottom ) . Tissues were harvested analyzed at day 7 after first tamoxifen injection . ( n = 5 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 reveals a progressive increase in YAP - TEAD target gene Ctgf in Lats1 / 2 dKO intestines at 3 - days 7 - days post i . p . injection with tamoxifen , as well as similarly increased TEAD1 TEAD4 expression , confirming that TEAD1 TEAD4 are YAP - responsive genes . Notably , Axin2 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 , 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 of TEAD1 / 2 / 4 mRNA expression from control ( wild - type ) ApcMin mutant tumours from small intestine ( top ) colon ( bottom ) . three genes were strongly induced within ApcMin mutant tumours , with TEAD2 being most strongly induced in colon ApcMin tumours ( n = 3 with multiple tumours within small 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 for TEAD4 reveals a gradient of expression along crypt - villus axis in both small 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 without affecting YAP subcellular localisation . of YAP is shown below , with statistical significance in an unpaired 2 - t - .

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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 from budding structures to cystic spheres increases YAP level . Scale bar is 250µm . n > 10 organoids in each experiment . of YAP from crypt villus regions of control organoids as well as from regions bounded by rectangles in Apc5 organoids are shown on right , with statistical significance in an unpaired 2 - t - .

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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.


A ) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP localisation after 4h of treatment in organoids cultured for 3d . n > 10 organoids in each experiment . B ) of of YAP per control crypt bud as shown in panel ( A ) , error bars show 1 SD ( * * * * p < 0 . 0001 ) . n > 10 organoids per condition ( top ) . of / of YAP per in Apc5 mutant organoids as shown in panel ( C ) . n > 10 organoids per condition ( bottom ) . Statistical was performed with an unpaired student ' s t - . C

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A) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d. n > 10 organoids in each experiment. B) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** p <0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired student's t-test. C


C ) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to cytoplasm . n > 10 organoids in each experiment . Q - PCR of YAP - TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 ( relative expression , error bars = 1 SD ) . Statistical was performed with an unpaired student ' s t - .

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C) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. Q-PCR analysis of the YAP-TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired student's t-test.


A ) Mouse intestinal organoids immunostained for YAP ( green ) . Note mostly localisation of YAP at 48h in organoid . Irradiation with 4Gy of X - rays drives strong localisation of YAP in many , which is reversible by treatment with Src family kinase inhibitor Dasatinib . Dasatinib treated organoids exhibit increased after irradiation , as indicated by pyknotic nuclei ( arrow ) . n > 10 organoids in each experiment . This experiment employs same control batch of 2 - 3d organoids also shown in Figure 6A .

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A) Mouse intestinal organoids immunostained for YAP (green). Note the mostly cytoplasmic localisation of YAP at 48h in the organoid. Irradiation with 4Gy of X-rays drives strong nuclear localisation of YAP in many cells, which is reversible by treatment with the Src family kinase inhibitor Dasatinib. The Dasatinib treated organoids exhibit increased cell death after irradiation, as indicated by pyknotic nuclei (white arrow). n > 10 organoids in each experiment. This experiment employs the same control batch of 2-3d organoids also shown in Figure 6A.


B ) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly localisation . Irradiation with 14Gy of X - rays drives strong localisation of YAP along entire crypt - villus axis . Deletion of Src within VillinCreERt Srcflox / flox intestines prevents YAP localisation normal regeneration after 14Gy irradiation . Arrows point to villar with YAP localisation . Lower panels show high - mag views of a different example than those shown in upper panels .

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B) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation. Irradiation with 14Gy of X-rays drives strong nuclear localisation of YAP along the entire crypt-villus axis. Deletion of Src within VillinCreERt Srcflox/flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation. Arrows point to villar cells with nuclear YAP localisation. Lower panels show high-mag views of a different example than those shown in upper panels.


A ) Control irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA ( black arrows point to mRNA granules in control ) by immunostaining for Sox9 reveals an expansion in expression domain of both genes . localisation of beta - catenin is readily detectable after irradiation . n = 3 samples per condition , shown in insets , error bars = 1 SD . Statistical was performed with an unpaired student ' s t - . Similar results were obtained at 3dpi 5dpi .

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A) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA (black arrows point to mRNA granules in the control) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes. Nuclear localisation of beta-catenin is readily detectable after irradiation. n = 3 samples per condition, quantification shown in the insets, error bars = 1 SD. Statistical analysis was performed with an unpaired student's t-test. Similar results were obtained at 3dpi and 5dpi.


B ) Q - PCR of YAP , TEADs their target genes Ctgf Cyr61 over a 4 days post irradiation time course at 12Gy . Note strong upregulation of TEAD4 , Ctgf , Cyr61 . In contrast , Axin2 , TEAD2 Sox9 mRNA expression are only moderately altered , confirming that Wnt signalling remains active upon irradiation - induced YAP - TEAD activation . n = 4 biological replicates per time point . Statistical was performed with unpaired t - compared to control 0Gy condition , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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B) Q-PCR analysis of YAP, TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy. Note the strong upregulation of TEAD4, Ctgf, and Cyr61. In contrast, Axin2, TEAD2 and Sox9 mRNA expression levels are only moderately altered, confirming that Wnt signalling remains active upon irradiation-induced YAP-TEAD activation. n = 4 biological replicates per time point. Statistical analysis was performed with unpaired t-test compared to the control 0Gy condition, * p<0.05, ** p<0.01, ***p<0.001.


C ) Control irradiated murine small intestines analysed by immunostaining for Sox9 reveals that treatment with Porcupine inhibitor LGK974 strongly reduces Sox9 expression domain induced by 3 days after 12Gy irradiation . Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 ( n = 4 in each condition ) .

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C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition).


D ) Q - PCR of Wnt YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation . Note inhibition of Axin2 , Sox9 , YAP , Ctgf , Cyr61 , TEAD family expression upon treatment with Porcupine inhibitor LGK974 . mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation ( 1dpi ) becomes more pronounced at 3 days after irradiation ( 3dpi ) . n = 4 biological replicates per condition , statistical was performed with Student ' s 2 - t - , error bars = 1 SD , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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D) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.


A . Volcano plots of genes that are differentially expressed in infected relative to mock - infected at 12 hpi 24 hpi in ileum organoids . statistical significance ( - log10 of adjusted p - value ) is shown as a function of log2 fold change . MAST were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in panels are gene names of selected differentially expressed genes between compared two populations .

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A. Volcano plots of genes that are differentially expressed in infected cells relative to mock-infected cells at 12 hpi and 24 hpi in ileum organoids. The statistical significance (-log10 of the adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations.


F . Multiplex in situ hybridization of ACE2 SARS - CoV - 2 of mock - infected infected 2D ileum organoids at 12 hpi 24 hpi . arrows point at SARS - CoV - 2 - infected . A representative image is shown .

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F. Multiplex in situ RNA hybridization of ACE2 and SARS-CoV-2 of mock-infected and infected 2D ileum organoids at 12 hpi and 24 hpi. White arrows point at SARS-CoV-2-infected cells. A representative image is shown.


Volcano plots displaying genes that are differentially expressed in immature enterocytes 2 upon SARS - CoV - 2 infection of colon organoids . infected vs . mock infected statistical significance ( - log10 adjusted p - value ) is shown as a function of log2 fold change . MAST were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in blue in panels are gene names of selected differentially expressed genes between compared two populations . Labeled dots in red in panels are gene corresponding to interferon if detected .

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

Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected.