Bright field captures of TDGwt , TDGcat and TDGsnm mESCs at indicated stages of differentiation . DAPI , Alexa Fluor ® 680 phalloidin actin staining at the bottom . Scale bars 25µM , 50µM , 500µM as indicated . Quantification of neural cells with neurites of total differentiating TDGwt , TDGcat and TDGsnm mESCs . n ≥ 5 . Means with standard deviation ( SD ) . * * : P < 0 . 01 . Non parametric , Mann - Whitney test .

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Bright field captures of TDGwt, TDGcat and TDGsnm mESCs at indicated stages of differentiation. DAPI, Alexa Fluor® 680 phalloidin actin staining at the bottom. Scale bars 25µM, 50µM, 500µM as indicated. Quantification of neural cells with neurites of total differentiating TDGwt, TDGcat and TDGsnm mESCs. n ≥ 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.


Relative changes of lineage marker expression between NPCs and undifferentiated mESCs ( ESCM LIF , 0 h ) , quantified with Gapdh as normalizer . n ≥ 3 . Means with standard deviation ( SD ) ) . * : P < 0 . 05 ; * * : P < 0 . 01 by non parametric Mann - Whitney test .

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Relative changes of lineage marker expression between NPCs and undifferentiated mESCs (ESCM LIF, 0 h), quantified with Gapdh as normalizer. n ≥ 3. Means with standard deviation (SD)). *: P<0.05; **: P<0.01 by non parametric Mann-Whitney test.


LC - MS / MS analysis of global cytosine - modification levels in DNA from ground - state mESCs ( 2i ) and EBs . Means with standard deviation ( SD ) of 5mC , 5hmC , 5fC and 5caC per 106 bases ) . n ≥ 3 . * : P ≤ 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 0001 by one - way ANOVA , Dunnett ' s multiple comparisons test .

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LC-MS/MS analysis of global cytosine-modification levels in DNA from ground-state mESCs (2i) and EBs. Means with standard deviation (SD) of 5mC, 5hmC, 5fC and 5caC per 106 bases). n ≥ 3. *: P≤0.05, **: P<0.01, ***: P<0.0001 by one-way ANOVA, Dunnett's multiple comparisons test.


Alkaline comet assay with pluripotent ( 0 h ) and differentiating ( 24 h 5 µM RA ) mESC as indicated . Olive Tail Moments and % DNA in tail . n ≥ 3 . Medians with standard deviation ( SD ) . * : P ≤ 0 . 05 ; * * : P ≤ 0 . 01 , * * * : P ≤ 0 . 0001 by one - way ANOVA , Sidak ' s multiple comparisons test .

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Alkaline comet assay with pluripotent (0 h) and differentiating (24 h 5 µM RA) mESC as indicated. Olive Tail Moments and % DNA in tail. n ≥ 3. Medians with standard deviation (SD). *: P≤0.05; **: P≤0.01, ***: P≤0.0001 by one-way ANOVA, Sidak's multiple comparisons test.


Cell proliferation assay . Relative viability of Talazoparib treated to untreated cells . n = 3 . Means with standard deviation ( SD ) , P ≤ 0 . 05 , * * : P < 0 . 01 , by one - way ANOVA , Dunnett ' s multiple comparisons test .

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Cell proliferation assay. Relative viability of Talazoparib treated to untreated cells. n = 3. Means with standard deviation (SD), P≤0.05, **: P<0.01, by one-way ANOVA, Dunnett's multiple comparisons test.


ChIP - qPCR analysis of TET1 , TDG and XRCC1 at gene promoters in mESCs ( 24 h 5 µM RA ) . Relative enrichments to an unbound control region ( Chr2neg ) . n ≥ 3 . Means with standard deviation ( SD ) . * : P ≤ 0 . 05 ; * * : P < 0 . 001 ; * * * : P ≤ 0 . 0001 , by two - tailed Mann - Whitney test .

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ChIP-qPCR analysis of TET1, TDG and XRCC1 at gene promoters in mESCs (24 h 5 µM RA). Relative enrichments to an unbound control region (Chr2neg). n ≥ 3. Means with standard deviation (SD). *: P≤0.05; **: P<0.001; ***: P≤0.0001, by two-tailed Mann-Whitney test.


Representative images of mESC neuronal differentiation as in Fig 4B . Shown are XRCC1wt and XRCC1null ( XRCC1null1 , XRCC1null2 ) mESCs in 2i medium supplemented with LIF ( 2i LIF ) and in ESC medium supplemented with LIF ( ESCM LIF ) , EBs in ESCM LIF and 5µM RA ( EB 4d ESCM RA ) and dissociated neuron - like cells in B27 ( B27 ) . Scale bars 25µM , 50µM , 500µM as indicated . Neurite grids were observed for XRCC1wt ; heterogeneous cell populations formed with the XRCC1null ( XRCC1null1 , null2 ) clones . Quantification ( below ) of neural cells with neurites of total differentiating XRCC1wt , XRCC1null mESCs . n = 5 . Means with standard deviation ( SD ) . * * : P < 0 . 01 . Non parametric , Mann - Whitney test .

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Representative images of mESC neuronal differentiation as in Fig 4B. Shown are XRCC1wt and XRCC1null (XRCC1null1, XRCC1null2) mESCs in 2i medium supplemented with LIF (2i LIF) and in ESC medium supplemented with LIF (ESCM LIF), EBs in ESCM LIF and 5µM RA (EB 4d ESCM RA) and dissociated neuron-like cells in B27 (B27). Scale bars 25µM, 50µM, 500µM as indicated. Neurite grids were observed for XRCC1wt; heterogeneous cell populations formed with the XRCC1null (XRCC1null1, null2) clones. Quantification (below) of neural cells with neurites of total differentiating XRCC1wt, XRCC1null mESCs. n = 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.


Quantification of mRNA expression of pluripotency and ectodermal / neurogenesis marker genes by RT - qPCR . n ≥ 3 . Mean values and standard deviations ( SD ) of EBs relative to their mESC ( ESCM LIF , 16h ) , normalised to Gapdh .

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Quantification of mRNA expression of pluripotency and ectodermal/neurogenesis marker genes by RT-qPCR. n ≥ 3. Mean values and standard deviations (SD) of EBs relative to their mESC (ESCM LIF, 16h), normalised to Gapdh.


( A ) Single confocal sections of HeLa cells infected with GFP - expressing S . Typhimurium strains ( blue ) for 10 h , fixed and immunolabelled for LAMP1 ( green ) and ubiquitin ( Ub , red ) ( false coloured , scale bars , 5 µm ) . The far right panels show merged images of LAMP1 , ubiquitin and Salmonella . Arrows indicate SCV - associated ubiquitinated aggregates .

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(A) Single confocal sections of HeLa cells infected with GFP-expressing S. Typhimurium strains (blue) for 10 h, fixed and immunolabelled for LAMP1 (green) and ubiquitin (Ub, red) (false coloured, scale bars, 5 µm). The far right panels show merged images of LAMP1, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitinated aggregates.


B ) Quantification of the percentage of infected cells containing SCV - associated ubiquitinated aggregates . Cells were processed as in ( A ) and analysed by fluorescence microscopy .

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B) Quantification of the percentage of infected cells containing SCV-associated ubiquitinated aggregates. Cells were processed as in (A) and analysed by fluorescence microscopy.


( C ) Kinetics of the formation of SCV - associated ubiquitinated aggregates . Cells were infected with GFP - expressing S . Typhimurium strains for the indicated amount of time , and processed as in ( A ) .

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(C) Kinetics of the formation of SCV-associated ubiquitinated aggregates. Cells were infected with GFP-expressing S. Typhimurium strains for the indicated amount of time, and processed as in (A).


( D ) Quantification of the percentage of infected cells with SCV - associated ubiquitinated aggregates in HeLa cells infected with S . Typhimurium strains for 14 h . Cells were processed as in ( A ) and analysed by fluorescence microscopy . For all quantifications , a minimum of 50 infected cells were counted per experiment and values are the mean ± SEM from at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(D) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates in HeLa cells infected with S. Typhimurium strains for 14 h. Cells were processed as in (A) and analysed by fluorescence microscopy. For all quantifications, a minimum of 50 infected cells were counted per experiment and values are the mean ± SEM from at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.


A ) Immunoelectron microscopy of HeLa cells infected with S . Typhimurium strains for 12 h ( ubiquitin - 10 nm gold particles ; scale bars , 0 . 5 µm ) . Arrows indicate ubiquitin accumulations near bacteria within vacuoles .

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A) Immunoelectron microscopy of HeLa cells infected with S. Typhimurium strains for 12 h (ubiquitin - 10 nm gold particles; scale bars, 0.5 µm). Arrows indicate ubiquitin accumulations near bacteria within vacuoles.


( B ) Single confocal sections of HeLa cells infected with GFP - expressing ΔsseL mutant bacteria ( blue ) for 10 h and immunolabelled for ubiquitin ( all chain types - Ub , red ) and lysine - 48 - linked ( K48 - Ub , green ) or lysine - 63 - linked ( K63 - Ub , green ) ubiquitin chains ( false coloured , scale bars , 5 µm ) . Arrows indicate SCV - associated ubiquitin and K63 - Ub labelling .

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(B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 10 h and immunolabelled for ubiquitin (all chain types - Ub, red) and lysine-48-linked (K48-Ub, green) or lysine-63-linked (K63-Ub, green) ubiquitin chains (false coloured, scale bars, 5 µm). Arrows indicate SCV-associated ubiquitin and K63-Ub labelling.


SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies . Single confocal sections of HeLa cells infected with GFP - expressing ΔsseL mutant bacteria ( blue ) for 12 h and immunolabelled for ubiquitin ( Ub , red ) and ( A ) p62 ( green ) or ( B ) LC3 ( green ) ( false coloured , scale bars , 5 µm ) . The far right panels show merged images of p62 or LC3 , ubiquitin and Salmonella . Arrows indicate SCV - associated ubiquitin labelling with p62 or LC3 .

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SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3.


( C - E ) . Quantification of the relative fluorescence intensity of ( C ) ubiquitin , ( D ) p62 and ( E ) LC3 per microcolony ( see Materials and Methods ) . Cells were processed as in ( A ) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment . Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown . Similar results were obtained in at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(C-E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.


( A ) Quantification of the percentage of infected cells with SCV - associated ubiquitinated aggregates . HeLa cells were infected with ΔsseL mutant bacteria for 12 h . At 8 h post - infection , cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling . A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments . p values are relative to the mean value of mock - treated cells . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001.


( B ) Single confocal sections of HeLa cells infected with GFP - expressing ΔsseL mutant bacteria ( blue ) , treated and processed as in ( A ) and analysed by fluorescence microscopy for ubiquitin ( Ub , red ) and LC3 ( green ) ( scale bars , 5 µm ) . The far right panels show merged images of LC3 , ubiquitin and Salmonella .

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(B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.


( A ) Single confocal sections of RAW264 . 7 macrophages infected with GFP - expressing strains of S . Typhimurium ( green ) for 10 h and immunolabelled for ubiquitin ( Ub , red ) ( scale bars , 5 µm ) .

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(A) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (green) for 10 h and immunolabelled for ubiquitin (Ub, red) (scale bars, 5 µm).


( B ) Quantification of infected macrophages containing ubiquitinated aggregates 10 h after bacterial uptake . Cells were processed as in ( A ) and analysed by fluorescence microscopy . A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of at least 3 independent experiments . * p < 0 . 05 ; * * * p < 0 . 001 . Uninfected cells ( uninf ) were from the same wells as infected and therefore were exposed to extracellular bacteria . ( C ) Immunoelectron microscopy of RAW264 . 7macrophages infected with the ΔsseL mutant bacteria for 12 h . Arrows and arrowheads indicate ubiquitin ( 15 nm gold particles ) and p62 ( 10 nm gold particles ) , respectively ( scale bar , 0 . 5 µm ) .

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(B) Quantification of infected macrophages containing ubiquitinated aggregates 10 h after bacterial uptake. Cells were processed as in (A) and analysed by fluorescence microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of at least 3 independent experiments. * p<0.05; *** p<0.001. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. (C) Immunoelectron microscopy of RAW264.7macrophages infected with the ΔsseL mutant bacteria for 12 h. Arrows and arrowheads indicate ubiquitin (15 nm gold particles) and p62 (10 nm gold particles), respectively (scale bar, 0.5 µm).


( D and E ) Single confocal sections of RAW264 . 7 macrophages infected with GFP - expressing strains of S . Typhimurium ( blue ) and immunolabelled for ubiquitin ( Ub , red ) and ( D ) p62 ( green ) or ( E ) LC3 ( green ) . The far right panels show merged images of p62 or LC3 , ubiquitin and Salmonella . Arrows indicate Arrows indicate SCV - associated ubiquitin labelling with p62 or LC3 ( scale bars , 5 µm ) . Cell outlines and nuclei are delineated by white lines .

(D and E) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (blue) and immunolabelled for ubiquitin (Ub, red) and (D) p62 (green) or (E) LC3 (green). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3 (scale bars, 5 µm). Cell outlines and nuclei are delineated by white lines.


( A ) Quantification of large ( > 2 µm ) , ubiquitinated aggresome - like induced structures ( ALIS ) in macrophages . Primary bone marrow - derived macrophages ( BMM ) were infected with the indicated GFP - expressing S . Typhimurium strains for 10 h , immunolabelled for ubiquitin and analysed by confocal microscopy . A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of 3 independent experiments . Uninfected cells ( uninf ) were from the same wells as infected and therefore were exposed to extracellular bacteria . * p < 0 . 05 ; * * p < 0 . 01 .

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(A) Quantification of large (>2 µm), ubiquitinated aggresome-like induced structures (ALIS) in macrophages. Primary bone marrow-derived macrophages (BMM) were infected with the indicated GFP-expressing S. Typhimurium strains for 10 h, immunolabelled for ubiquitin and analysed by confocal microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of 3 independent experiments. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. * p<0.05; ** p<0.01.


( B ) Quantification of the number of puromycin - induced ubiquitin inclusions in cells . HeLa cells were transfected with a vector expressing myc - SseL or myc - SseLC / A for 16 h followed by treatment with puromycin ( 5 µg / ml ) for 4 h and immunolabelled with anti - myc , anti - p62 and anti - ubiquitin . 50 individual cells were counted per condition in each experiment . Values are the means ± SEM of at least 3 independent experiments . * * p < 0 . 01 .

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(B) Quantification of the number of puromycin-induced ubiquitin inclusions in cells. HeLa cells were transfected with a vector expressing myc-SseL or myc-SseLC/A for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc, anti-p62 and anti-ubiquitin. 50 individual cells were counted per condition in each experiment. Values are the means ± SEM of at least 3 independent experiments. ** p<0.01.


( C ) Single confocal sections of HeLa cells transfected with a vector expressing myc - SseL for 16 h followed by treatment with puromycin ( 5 µg / ml ) for 4 h and immunolabelled with anti - myc ( blue ) , anti - p62 ( green ) and anti - ubiquitin ( Ub , red ) ( scale bars , 5 µm ) . The lower panels show a merged image of p62 , ubiquitin and myc - SseL . Arrows indicate ubiquitin and p62 aggregates present in untransfected cells and arrowheads indicate cells expressing myc -

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(C) Single confocal sections of HeLa cells transfected with a vector expressing myc-SseL for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc (blue), anti-p62 (green) and anti-ubiquitin (Ub, red) (scale bars, 5 µm). The lower panels show a merged image of p62, ubiquitin and myc-SseL. Arrows indicate ubiquitin and p62 aggregates present in untransfected cells and arrowheads indicate cells expressing myc-SseL


SseL interacts with p62 and autophagic substrates . ( A and B ) Co - immunoprecipitation from RAW264 . 7 macrophages infected with ΔsseL mutant bacteria expressing SseL - HA or SseLC / A - HA for 10 h . ( A ) Infectedcell lysates and immunoprecipated fractions were probed with anti - HA , anti - ubiquitin and anti - p62 antibodies . ( B ) At 7 h post - infection , infected cells were subjected to mock , 3 - MA or starvation ( Stv ) treatments for 3 h before harvesting and processing as described in ( A ) . Cell lysates and immunoprecipiations were probed with anti - HA and anti - ubiquitin antibodies .

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SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.


( A ) Lipidated LC3B ( LC3 - II ) levels in RAW264 . 7 macrophages infected with the indicated strains of S . Typhimurium for 12 h . Cells were left untreated ( − ) or subjected to 10 mM NH4Cl treatment ( + ) for 2 h before harvesting .

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(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting.


( B ) Quantification of relative LC3 II levels in infected cells . Denistometry of bands was analysed using Image J software . The ratio of LC3 - II / tubulin signal intensity was normalized to 1 for wild - type untreated infections . The graph shows the mean relative LC3 II / tubulin ratios ± SEM for 3 independent experiments .

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(B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments.


C ) RAW264 . 7 or ( D ) murine bone marrow - derived primary macrophages were infected with the indicated strains of S . Typhimurium for ( C ) 10 h or ( D ) 16 h and bacterial replication was monitored using flow cytometry . Results are represented as fold replication normalized to wt ; values are the means ± SEM of at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 .

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C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.


a , Seckel cells contain normal centrosome numbers and mitotic spindle poles . Interphase centrosomes of Seckel cells have no CPAP immunoreactivity but recruit similar levels of centrosomal proteins ( ii and iii ) .

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a, Seckel cells contain normal centrosome numbers and mitotic spindle poles. Interphase centrosomes of Seckel cells have no CPAP immunoreactivity but recruit similar levels of centrosomal proteins (ii and iii).


b , Seckel cells display overly long cilia . Arl13b labels cilium . Scale bar 1 μm . Quantification of cilia length , * * * p < 0 . 001 ; t - test ; Scatter plot showing mean with ± SD ( iii ) . Cells displaying normal ( 0 - 5 μm ) , long ( 5 - 10 μm ) and extra long ( > 10 μm ) cilia . * * * p < 0 . 001 ; Ordinary Two - way ANOVA ( iv ) . Average cilia length of WT and Seckel cells ( v ) . n = 3 independent experiments , 228 cells for WT and 297 cells for Seckel . Error bars are s . e . m . * * * p < 0 . 001 , t - test .

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b, Seckel cells display overly long cilia. Arl13b labels cilium. Scale bar 1 μm. Quantification of cilia length, *** p<0.001; t-test; Scatter plot showing mean with ±SD (iii). Cells displaying normal (0-5 μm), long (5-10 μm) and extra long (>10 μm) cilia. *** p<0.001; Ordinary Two-way ANOVA (iv). Average cilia length of WT and Seckel cells (v). n = 3 independent experiments, 228 cells for WT and 297 cells for Seckel. Error bars are s. e. m. *** p<0.001, t-test.


c , Electron microscopy ( EM ) of Seckel and WT fibroblasts . In contrast to WT , Seckel basal body has disorganized microtubule of doublet and triplets ( red arrows and red circles in schematics ) . d , Longitudinal serial - sectioning EM of WT ( top ) Seckel fibroblasts ( bottom ) . In contrast to WT basal bodies that have structurally normal distal and sub - distal appendages ( yellow arrows and red triangles of DA and SDA in schematics ) , Seckel basal body displays either lack or misplaced appendages . n > 15 basal bodies . e , Seckel cells show reduced EdU incorporation after 72 hrs . Error bars are s . e . m . * * p < 0 . 01 , ttest ; n = 4 independent experiments , 727 cells for WT and 844 cells for Seckel .

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c, Electron microscopy (EM) of Seckel and WT fibroblasts. In contrast to WT, Seckel basal body has disorganized microtubule of doublet and triplets (red arrows and red circles in schematics).d, Longitudinal serial-sectioning EM of WT (top) Seckel fibroblasts (bottom). In contrast to WT basal bodies that have structurally normal distal and sub-distal appendages (yellow arrows and red triangles of DA and SDA in schematics), Seckel basal body displays either lack or misplaced appendages. n> 15 basal bodies.e, Seckel cells show reduced EdU incorporation after 72 hrs. Error bars are s. e. m. ** p<0.01, ttest; n = 4 independent experiments, 727 cells for WT and 844 cells for Seckel.


a . Experimental plan . Both Seckel and WT cells were synchronized at G0 by serum starvation for 96 hrs . This treatment causes Seckel and WT cells to have equivalent cilia length allowing us to estimate the rate of cilia disassembly ( see below and Appendix Fig . S2a ) . b , Dynamic CPAP localization during cilium disassembly and cell cycle re - entry . Cep152 levels remain unchanged . Arl13b ( green ) or acetylated α - tubulin ( magenta ) marks the cilium . n > 100 cells . Bar diagrams at right shows semi quantitative intensity measurements . Error bars are ± SD .

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a. Experimental plan. Both Seckel and WT cells were synchronized at G0 by serum starvation for 96 hrs. This treatment causes Seckel and WT cells to have equivalent cilia length allowing us to estimate the rate of cilia disassembly (see below and Appendix Fig. S2a).b, Dynamic CPAP localization during cilium disassembly and cell cycle re-entry. Cep152 levels remain unchanged. Arl13b (green) or acetylated α-tubulin (magenta) marks the cilium. n> 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.


c , Seckel cells show neither CPAP dynamics nor cilium disassembly upon serum stimulation . Cep152 levels remain constant . n > 100 cells . Bar diagrams at right shows semi quantitative intensity measurements . Error bars are ± SD .

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c, Seckel cells show neither CPAP dynamics nor cilium disassembly upon serum stimulation. Cep152 levels remain constant. n> 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.


d , Cilium disassembly dynamics in WT and Seckel cells . Relative cilium disassembly rate shows retarded cilium disassembly in Seckel cells ( Error bars are s . e . m . ) ( ii ) . Quantification and distribution of cilia length in WT and Seckel fibroblasts . Scatter plot shows mean with ± SD ( i ) .

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d, Cilium disassembly dynamics in WT and Seckel cells. Relative cilium disassembly rate shows retarded cilium disassembly in Seckel cells (Error bars are s. e. m.) (ii). Quantification and distribution of cilia length in WT and Seckel fibroblasts. Scatter plot shows mean with ± SD (i).


e , Seckel cells with retarded cilium disassembly show delayed G1 - S transition assessed by nuclearCyclin A immunoreactivity . n = 3 independent experiments , n > 200 cells for WT and Seckel . Error bars are s . e . m .

e, Seckel cells with retarded cilium disassembly show delayed G1-S transition assessed by nuclearCyclin A immunoreactivity. n = 3 independent experiments, n> 200 cells for WT and Seckel. Error bars are s.e.m.


f , Seckel cells show delayed mitotic entry . Note that an unresorbed Arl13b - positive ciliary remnant ( ciliary membrane , green ) is present with one of the centrosomes ( Paridaen et al , 2013 ) . Bar diagram shows mitotic index of WT and Seckel cells 24 hrs after serum stimulation . n = 3 independent experiments , n > 300 cells for WT and Seckel . Error bars are s . e . m . Scale bar 1 μm .

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f, Seckel cells show delayed mitotic entry. Note that an unresorbed Arl13b-positive ciliary remnant (ciliary membrane, green) is present with one of the centrosomes(Paridaen et al, 2013). Bar diagram shows mitotic index of WT and Seckel cells 24 hrs after serum stimulation. n = 3 independent experiments, n> 300 cells for WT and Seckel. Error bars are s.e.m. Scale bar 1 μm.


a , CPAP co - immunopurifies with CDC components from HeLa extracts . Centrosomal proteins Cap350 and ODF2 do not co - purify with CPAP .

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a, CPAP co-immunopurifies with CDC components from HeLa extracts. Centrosomal proteins Cap350 and ODF2 do not co-purify with CPAP.


b , Levels of CDC components ( CPAP , Aurora A , OFD1 , and Nde - 1 ) are differentially regulated in response to serum starvation and stimulation in WT and Seckel fibroblasts . Specifically , they are elevated 1h after serum stimulation , thus enabling cilium disassembly . Elevated CDC levels in WT cells correlate with an elevated level of CPAP as shown in Fig . 2 as well as with the expression of cyclin A , a G1 - S marker . GAPDH was used as a loading control indicating an equivalent amount of WT and Seckel fibroblast total proteins were loaded .

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b, Levels of CDC components (CPAP, Aurora A, OFD1, and Nde-1) are differentially regulated in response to serum starvation and stimulation in WT and Seckel fibroblasts. Specifically, they are elevated 1h after serum stimulation, thus enabling cilium disassembly. Elevated CDC levels in WT cells correlate with an elevated level of CPAP as shown in Fig. 2 as well as with the expression of cyclin A, a G1-S marker. GAPDH was used as a loading control indicating an equivalent amount of WT and Seckel fibroblast total proteins were loaded.


c . WT CPAP ( WT - cells IP ) but not Seckel variant CPAP ( Seckel cells IP ) co - immunopurifies with CDC components from asynchronously grown WT and Seckelfibroblastscell extracts . The IP samples were adjusted so that equivalent amounts of immunoprecipitatedCPAP were used in samples . Seckel ( 5x ) and WT ( 1x ) fibroblast cell extracts were used as input controls to show the efficacy of the various antibodies . Note that CPAP abundance in Seckel cells is ~ 5 times lower than that of WT cells . CentrosomalproteinsCap350 and ODF2 do not co - purify with CPAP indicating the specificity of CPAP interaction with CDC components .

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c. WT CPAP (WT-cells IP) but not Seckel variant CPAP (Seckel cells IP) co-immunopurifies with CDC components from asynchronously grown WT and Seckelfibroblastscell extracts. The IP samples were adjusted so that equivalent amounts of immunoprecipitatedCPAP were used in samples. Seckel (5x) and WT (1x) fibroblast cell extracts were used as input controls to show the efficacy of the various antibodies. Note that CPAP abundance in Seckel cells is ~5 times lower than that of WT cells. CentrosomalproteinsCap350 and ODF2 do not co-purify with CPAP indicating the specificity of CPAP interaction with CDC components.


d , GST pull down ( i ) and immunopurification assays ( ii ) of CPAP ' s CC5 domain demonstrate its sufficiency in mediating CPAP - CDC protein interactions . The CC5 fragment is expressed as a GFP fusion protein in HeLa cells and immunopurified using anti - GFP antibodies . HeLa extracts were used as input controls . CAP350 does not co - purify with CPAP . Both Cap350 and ODF2 are not pulled down by CPAP ' s CC5 domain .

d, GST pull down (i) and immunopurification assays (ii) of CPAP's CC5 domain demonstrate its sufficiency in mediating CPAP-CDC protein interactions. The CC5 fragment is expressed as a GFP fusion protein in HeLa cells and immunopurified using anti-GFP antibodies. HeLa extracts were used as input controls. CAP350 does not co-purify with CPAP. Both Cap350 and ODF2 are not pulled down by CPAP's CC5 domain.


a , Seckel NPCs fail to efficiently recruit CDC components . Fraction of non - ciliated and ciliated WT and Seckel NPCs . Ciliated cells are further quantified and grouped based on whether they are positive or negative for Aurora A and CPAP at their basal bodies . Scale bar 0 . 5 μm ( i - ii ) . Proportion of ciliated cells is higher in Seckel NPCs ( * * p < 0 . 01 ) . Compared to WT , a higher proportion of Seckel NPCs lack Aurora A and CPAP in their basal bodies ( * p < 0 . 05 for Auroa A and * * * p < 0 . 001 for CPAP ) . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 3 independent experiments . 127 cells for WT and 97 cells for Seckel . Error bars are s . e . m . ( iii ) Seckel cells consistently contain Nde - 1 and Ofd1 with a significantly lesser intensity . t test ; error bars are s . e . m . ; n = 3 independent experiments . 110 cells for WT and 100 cells for Seckel .

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a, Seckel NPCs fail to efficiently recruit CDC components. Fraction of non-ciliated and ciliated WT and Seckel NPCs. Ciliated cells are further quantified and grouped based on whether they are positive or negative for Aurora A and CPAP at their basal bodies. Scale bar 0.5 μm (i-ii). Proportion of ciliated cells is higher in Seckel NPCs (**p<0.01). Compared to WT, a higher proportion of Seckel NPCs lack Aurora A and CPAP in their basal bodies (*p<0.05 for Auroa A and ***p<0.001 for CPAP). Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel. Error bars are s. e. m. (iii) Seckel cells consistently contain Nde-1 and Ofd1 with a significantly lesser intensity. t test; error bars are s.e.m.; n = 3 independent experiments. 110 cells for WT and 100 cells for Seckel.


b , Proportion of ciliated cells is higher in Seckel NPCs . Error bars are s . e . m . * * p < 0 . 01 , t - test . n = 3 independent experiments . 127 cells for WT and 97 cells for Seckel .

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b, Proportion of ciliated cells is higher in Seckel NPCs. Error bars are s. e. m. ** p<0.01, t-test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel.


c , Seckel NPCs exhibit longer cilia . Error bars are ± s . e . m . * * * p < 0 . 001 , t - test . NPCs were derived from two independent iPS clones . n = 3 independent experiments . 146 cells for WT , 135 cells for Seckel ( clone 1 ) and 202 ( clone 2 ) . d , SeckelNPCs show reduced proliferation and delayed G1 - S transition . Error bars are ± s . e . m . * * * p < 0 . 001 ( EdU ) , * p < 0 . 05 ( Cyclin - A ) , t - test . For cyclin A , n = 3 independent experiments . 270 cells for WT and 232 cells for Seckel cells . For EdU , 700 cells for WT and 1630 cells for Seckel .

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c, Seckel NPCs exhibit longer cilia. Error bars are ± s. e. m. *** p<0.001, t-test. NPCs were derived from two independent iPS clones. n = 3 independent experiments. 146 cells for WT, 135 cells for Seckel (clone 1) and 202 (clone 2).d, SeckelNPCs show reduced proliferation and delayed G1-S transition. Error bars are ± s. e. m. *** p<0.001 (EdU), * p<0.05 (Cyclin-A), t-test. For cyclin A, n = 3 independent experiments. 270 cells for WT and 232 cells for Seckel cells. For EdU, 700 cells for WT and 1630 cells for Seckel.


e , Olomoucine triggers differentiation of WT NPCs into TUJ1 - positive neurons under non - differentiating conditions . Error bars are s . e . m . * * * p < 0 . 001 , Ordinary Two - way ANOVA followed by Tukey ' s multiple comparisons test . n = 3 independent experiments . 298 cells for untreated , 151 cells for DMSO and 146 cells for olomoucine , 25 μM .

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e, Olomoucine triggers differentiation of WT NPCs into TUJ1-positive neurons under non-differentiating conditions. Error bars are s. e. m. *** p<0.001, Ordinary Two-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments. 298 cells for untreated, 151 cells for DMSO and 146 cells for olomoucine, 25 μM.


f , Seckel NPCs differentiated into TUJ1 - positive neurons under non - differentiating conditions . Error bars show ± s . e . m . * * * p < 0 . 001 , t - test . n = 3 independent experiments . 633 cells for WT and 275 cells for Seckel . Scale bar 5 μm .

f, Seckel NPCs differentiated into TUJ1-positive neurons under non-differentiating conditions. Error bars show ±s. e. m. *** p<0.001, t-test. n = 3 independent experiments. 633 cells for WT and 275 cells for Seckel. Scale bar 5 μm.


g , Expression of CPAP - GFP or CPAP - CC5 - GFP in Seckel NPCs slightly increases nestin - positive cells ( i ) , decreases neuronal differentiation ( for CC5 * p < 0 . 05 ) ( ii ) , decreases ciliary length ( for CPAP * * * p < 0 . 001 and for CC5 * * p < 0 . 01 ) ( iii ) , elevates mitotic index ( for CC5 * p < 0 . 05 ) ( iv ) and decreases the number of ciliated cells ( for CPAP * p < 0 . 05 and for CC5 * * p < 0 . 01 ) ( v ) compared to control ( mock electroporation ) . Error bars show s . e . m . One - way ANOVA followed by Tukey ' s multiple comparisons test . n = 3 independent experiments with > 350 ( nestinstaining , differentiation and mitotic index ) , and > 70 cells ( cilium length ) cells .

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g, Expression of CPAP-GFP or CPAP-CC5-GFP in Seckel NPCs slightly increases nestin-positive cells (i), decreases neuronal differentiation (for CC5 *p<0.05) (ii), decreases ciliary length (for CPAP ***p<0.001 and for CC5 **p<0.01) (iii), elevates mitotic index (for CC5 *p<0.05) (iv) and decreases the number of ciliated cells (for CPAP *p<0.05 and for CC5 **p<0.01) (v) compared to control (mock electroporation). Error bars show s. e. m. One-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments with >350 (nestinstaining, differentiation and mitotic index), and >70 cells (cilium length) cells.


a - b , siRNA - mediated down regulation of CDC components in WT NPCs causes longer cilia ( Ofd1 * * * p < 0 . 001 , Nde1 * * p < 0 . 01 ) , retarded cilia disassembly as shown by increased numbers of ciliated cells ( Ofd1 * * p < 0 . 01 , Nde1 * p < 0 . 05 ) and differentiation of NPCs into TUJ1 positive cells ( Ofd1 * * p < 0 . 001 , Nde1 * p < 0 . 01 ) . In controls , a non - targeting siRNA ( scrambled ) is used . Western blots at right validate siRNAs - mediated Nde1 and OFD1 depletion . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 3 independent experiments . 275 cells for scrambled , 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA .

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a-b, siRNA-mediated down regulation of CDC components in WT NPCs causes longer cilia (Ofd1 ***p<0.001, Nde1 **p<0.01), retarded cilia disassembly as shown by increased numbers of ciliated cells (Ofd1 **p<0.01, Nde1 *p<0.05) and differentiation of NPCs into TUJ1 positive cells (Ofd1 **p<0.001, Nde1 *p<0.01). In controls, a non-targeting siRNA (scrambled) is used. Western blots at right validate siRNAs-mediated Nde1 and OFD1 depletion. Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 275 cells for scrambled, 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA.


c , VX680 ( 0 . 5 μM , a concentration that will specifically allow neuronal differentiation ) was used to inhibit Aurora A activity . Bar diagrams below show quantifications . Compared to DMSO - treated control NPCs , a higher proportion of VX680 treated NPCs are ciliated ( * * * p < 0 . 001 ) , exhibiting longer cilia ( * * * p < 0 . 001 ) and show Tuj1 positive neurons ( * * * p < 0 . 001 ) . Error bars are s . e . m . For ciliated cells and cilium length : n = 3 independent experiments . 164 cells for DMSO - treated and 77 cells for VX680 - treated . For NPC differentiation : n = 4 independent experiments . 286 cells for DMSO - treated and 118 cells for VX680 - treated . Scale bar 1 μm .

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c, VX680 (0.5 μM, a concentration that will specifically allow neuronal differentiation) was used to inhibit Aurora A activity. Bar diagrams below show quantifications. Compared to DMSO-treated control NPCs, a higher proportion of VX680 treated NPCs are ciliated (***p<0.001), exhibiting longer cilia (***p<0.001) and show Tuj1 positive neurons (***p<0.001). Error bars are s. e. m. For ciliated cells and cilium length: n = 3 independent experiments. 164 cells for DMSO-treated and 77 cells for VX680-treated. For NPC differentiation: n = 4 independent experiments. 286 cells for DMSO-treated and 118 cells for VX680-treated. Scale bar 1 μm.


d , Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours . Inhibition of Ofd1 ( * p < 0 . 05 ) , Nde1 ( * * p < 0 . 01 ) and Aurora A ( * * * p < 0 . 001 ) caused significantly reduced incorporation of EdU .

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d, Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours. Inhibition of Ofd1 (*p<0.05), Nde1 (**p<0.01) and Aurora A (***p<0.001) caused significantly reduced incorporation of EdU.


e - g , Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP . Both low - and high - exposures are given ( e ) . Increased cilia length and NPC differentiation upon expression of RNAi - resistant CPAP E1235V compared to WT CPAP ( f - g ) . Error bars are s . e . m . * * * p < 0 . 001 ( cilia length ) , * p < 0 . 05 ( differentiation ) , t - test . For cilia length : n = 4 independent experiments . 25 cells for CPAP , 47 cells for CPAP E1235V . For differentiation : n = 4 independent experiments . 145 for CPAP and 81 for CPAP E1235V . Scale bar 1 μm .

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e-g, Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP. Both low -and high -exposures are given (e). Increased cilia length and NPC differentiation upon expression of RNAi-resistant CPAP E1235V compared to WT CPAP (f-g). Error bars are s. e. m. *** p<0.001 (cilia length), * p<0.05 (differentiation), t-test. For cilia length: n = 4 independent experiments. 25 cells for CPAP, 47 cells for CPAP E1235V. For differentiation: n = 4 independent experiments. 145 for CPAP and 81 for CPAP E1235V. Scale bar 1 μm.


a , iPS - derived 14 - day old organoid displaying complex morphological structures of brain - like tissues , parenthesis . Scale bar 500 μm . The ventricular zone ( VZ ) with radial glial cells stained with nestin ( magenta ) and neurons stained with TUJ1 is shown at higher magnification . Scale bar 20 μm .

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a, iPS-derived 14-day old organoid displaying complex morphological structures of brain-like tissues, parenthesis. Scale bar 500 μm. The ventricular zone (VZ) with radial glial cells stained with nestin (magenta) and neurons stained with TUJ1 is shown at higher magnification. Scale bar 20 μm.


b , Reduced Nestin / Pax - 6 - positive progenitors and increased TUJ1 - positive neurons in Seckel organoids . In ventricular zones of WT organoids , there are significantly more Pax6 positive cells than TUJ1 positive cells , which is not the case in Seckel organoids ( * p < 0 . 05 ) . TUJ1 / Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs . Pax6 cells in Seckel organoid ventricular zones ( * p < 0 . 05 ) . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons test . n = 10 WT organoids ( 2520 cells ) . Seckeln = 10 organoids ( 2024 cells ) . Scale bar 20 μm .

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b, Reduced Nestin/Pax-6-positive progenitors and increased TUJ1-positive neurons in Seckel organoids. In ventricular zones of WT organoids, there are significantly more Pax6 positive cells than TUJ1 positive cells, which is not the case in Seckel organoids (*p<0.05). TUJ1/Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs. Pax6 cells in Seckel organoid ventricular zones (* p<0.05). Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 10 WT organoids (2520 cells). Seckeln = 10 organoids (2024 cells). Scale bar 20 μm.


c , ( i ) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen ( * p < 0 . 05 ) . Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids . ( * p < 0 . 05 ) . Error bars are s . e . m . t - test . n = 6 for WT organoid and 6 for Seckel organoid . Scale bar 20 μm . ( ii ) Seckel organoid cells display longer cilia . Error bars are s . e . m . * * * p < 0 . 001 , t - test . n = 3 for WT organoids and 4 for Seckel organoids . Scale bar 1 μm .

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

c, (i) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen (* p<0.05). Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids. (*p<0.05). Error bars are s. e. m. t-test. n=6 for WT organoid and 6 for Seckel organoid. Scale bar 20 μm. (ii) Seckel organoid cells display longer cilia. Error bars are s. e. m. *** p<0.001, t-test. n=3 for WT organoids and 4 for Seckel organoids. Scale bar 1 μm.


d , Seckel organoids electroporated with constructs expressing GFP alone ( i ) , GFP with CC5 - GFP and ( ii ) GFP with CPAP - GFP ( iii ) . Middle panel shows staining with anti - GFP antibodies ( red ) to show the transfected area . Note the presence of increased Pax6 - positive ( magenta ) NPCs in CC5 and CPAP electroporated tissues compared to GFP control . Likewise , there is a larger neural epithelium with multiple RGs ( arrowheads ) . Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone ( iv ) lower TUJ1 / Pax6 ratio ( * * p < 0 . 01 and * * * p < 0 . 001 respectively ) ( v ) smaller lumen diameter ( * * p < 0 . 01 ) and ( vi ) shorter cilia length ( * * p < 0 . 01 and * * * p < 0 . 001 respectively ) . Error bars are s . e . m . One - way ANOVA followed by Dunnett ' s multiple comparisons test . n = 3 organoids with 225 cells for GFP , 537 cells for CPAP and 569 for CC5 . At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter . t test . Error bars are s . e . m . Scale bar 20 μm . e , Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho - vimentin - positive RGs in Seckel organoids . Compared to GFP control , electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs ( for CC5 * * * p < 0 . 001 ) and decreases the portion of obliquely ( for CC5 * p < 0 . 05 ) or vertically oriented SeckelGFP ( for CPAP and CC5 * * p < 0 . 01 ) . Error bars are s . e . m . Two - way ANOVA followed by Tukey ' s multiple comparisons test . n = 4 organoids . 100 cells for GFP control , 112 cells for CPAP and 99 cells for CC5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Seckel organoids electroporated with constructs expressing GFP alone (i), GFP with CC5-GFP and (ii) GFP with CPAP-GFP (iii). Middle panel shows staining with anti-GFP antibodies (red) to show the transfected area. Note the presence of increased Pax6-positive (magenta) NPCs in CC5 and CPAP electroporated tissues compared to GFP control. Likewise, there is a larger neural epithelium with multiple RGs (arrowheads). Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone (iv) lower TUJ1/Pax6 ratio (**p<0.01 and ***p<0.001 respectively) (v) smaller lumen diameter (**p<0.01) and (vi) shorter cilia length (**p<0.01 and ***p<0.001 respectively). Error bars are s. e. m. One-way ANOVA followed by Dunnett's multiple comparisons test. n = 3 organoids with 225 cells for GFP, 537 cells for CPAP and 569 for CC5. At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter. t test. Error bars are s.e.m. Scale bar 20 μm.e, Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho-vimentin-positive RGs in Seckel organoids. Compared to GFP control, electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs (for CC5 ***p<0.001) and decreases the portion of obliquely (for CC5 *p<0.05) or vertically oriented SeckelGFP (for CPAP and CC5 **p<0.01). Error bars are s.e.m. Two-way ANOVA followed by Tukey's multiple comparisons test. n = 4 organoids. 100 cells for GFP control, 112 cells for CPAP and 99 cells for CC5.


( B ) Changes in plasma level of each cocktail substances in both supplementation and control studies ( NR is detected in control study ) compared to time baseline based on untargeted metabolomics measurement . The grey shaded area represents the 95 % confidence level interval . For boxplots limits , the middle line represents the median . The upper and lower box limits represent the 25 % quantiles . The upper and lower error bars correspond to 75 % quantiles . The p - values are derived from one - way ANOVA ( FDR < 0 . 05 ) .

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

(B) Changes in plasma level of each cocktail substances in both supplementation and control studies (NR is detected in control study) compared to time baseline based on untargeted metabolomics measurement. The grey shaded area represents the 95% confidence level interval. For boxplots limits, the middle line represents the median. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. The p-values are derived from one-way ANOVA (FDR< 0.05).


( A - G ) Changes in plasma level of key metabolites compared to time baseline ( solid line ) based on untargeted metabolomics measurement in metabolic cofactor supplementation study . The upper and lower box limits represent the 25 % quantiles . The upper and lower error bars correspond to 75 % quantiles . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - G) Changes in plasma level of key metabolites compared to time baseline (solid line) based on untargeted metabolomics measurement in metabolic cofactor supplementation study. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. * Denotes significance (one-way ANOVA; FDR <0 .05).


( A ) A summary of significantly different plasma metabolites compared to time baseline in metabolic cofactor supplementation and control studies based on untargeted metabolomics measurements . Red and blue colors denote the increased and decreased plasma levels compared to baseline , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A summary of significantly different plasma metabolites compared to time baseline in metabolic cofactor supplementation and control studies based on untargeted metabolomics measurements. Red and blue colors denote the increased and decreased plasma levels compared to baseline, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( B ) Metabolites significantly different between supplementation and control studies . Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

(B) Metabolites significantly different between supplementation and control studies. Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( C - E ) Changes in plasma level of key metabolites compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies . The grey shaded area represents the 95 % confidence level interval .

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

(C-E) Changes in plasma level of key metabolites compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies. The grey shaded area represents the 95% confidence level interval.


( A ) A summary of significantly different plasma inflammatory protein markers compared to time baseline in metabolic cofactor supplementation and control studies . Red and blue colors denote the increased and decreased plasma levels compared to baseline , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A summary of significantly different plasma inflammatory protein markers compared to time baseline in metabolic cofactor supplementation and control studies. Red and blue colors denote the increased and decreased plasma levels compared to baseline, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( B ) Proteins significantly different between supplementation and control studies . Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proteins significantly different between supplementation and control studies. Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( C - F ) Changes in the plasma level of key proteins compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies . The grey shaded area represents the 95 % confidence level interval . For boxplots limits , the middle line represents the median . The upper and lower box limits represent the 25 % quantiles . The upper and lower error bars correspond to 75 % quantiles . The p - values are derived from one - way ANOVA ( FDR < 0 . 05 ) .

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

(C-F) Changes in the plasma level of key proteins compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies. The grey shaded area represents the 95% confidence level interval. For boxplots limits, the middle line represents the median. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. The p-values are derived from one-way ANOVA (FDR< 0.05).


( A ) Plasma serine interpolations for each of the nine subjects .

(A) Plasma serine interpolations for each of the nine subjects.


( B ) Model fit to the target concentration curve ( mean interpolation of plasma serine over 24 hours ) with standard deviation indicated in gray .

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

(B) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray.


( D ) Plasma L - carnitine interpolations for each of the nine subjects .

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

(D) Plasma L-carnitine interpolations for each of the nine subjects.


( E ) Model fit to the target concentration curve ( mean interpolation of plasma L - carnitine over 24 hours ) with standard deviation indicated in gray .

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

(E) Model fit to the target concentration curve (mean interpolation of plasma L-carnitine over 24 hours) with standard deviation indicated in gray.


( G ) Plasma NAC interpolations for each of the nine subjects .

(G) Plasma NAC interpolations for each of the nine subjects.


( H ) Model fit to the target concentration curve ( mean interpolation of plasma serine over 24 hours ) with standard deviation indicated in gray .

O O O 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) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray.


Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( A ) DSBs quantified by qPCR analysis of Igk ( Jκ1 ) genomic DNA . Schematic shows germline ( GL ) Igk locus , unrepaired Jκ1 coding end ( Cut ) and primer location . Results are normalized to Rag1 - / - : Bcl2 abl pre - B cells , which do not generate RAG DSBs and have only germline Igk DNA . Loss of Igk germline product is representative of DSB generation .

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

Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (A) DSBs quantified by qPCR analysis of Igk (Jκ1) genomic DNA. Schematic shows germline (GL) Igk locus, unrepaired Jκ1 coding end (Cut) and primer location. Results are normalized to Rag1-/-:Bcl2 abl pre-B cells, which do not generate RAG DSBs and have only germline Igk DNA. Loss of Igk germline product is representative of DSB generation.


Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( B ) Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified .

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

Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (B) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( C ) Western blot analysis of KAP1 phosphorylation ( p - KAP1 ) , NF - κB2 ( p100 and p52 ) and PIM2 . GAPDH is shown as loading control .

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

Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (C) Western blot analysis of KAP1 phosphorylation (p-KAP1), NF-κB2 (p100 and p52) and PIM2. GAPDH is shown as loading control.


Rag1 - / - : Bcl2 and Art - / - : Bcl2 abl pre - B cells were treated with imatinib for 48 h to induce G1 arrest . ( D ) Cd40 and Pim2 mRNA expression .

Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (D) Cd40 and Pim2 mRNA expression.


Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( E ) DSBs quantified by qPCR analysis of Igh ( JH1 ) genomic DNA and analyzed as in A with results normalized to empty vector control , which has only germline Igh DNA .

Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (E) DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in A with results normalized to empty vector control, which has only germline Igh DNA.


Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( F ) Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified .

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

Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (F) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


E - H . Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( G ) Western blot analysis of FLAG - RAG1 , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H. Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (G) Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.


Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( H ) Cd40 and Pim2 mRNA expression .

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

Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (H) Cd40 and Pim2 mRNA expression.


A . Rag1 - / - : Lig4 - / - : Bcl2 : iCas9 abl pre - B cells were treated with ( + ) or without ( - ) 2 μM doxycycline ( Dox ) to induce Cas9 as indicated . Western blot shows FLAG - Cas9 and GAPDH ( loading control ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with (+) or without (-) 2 μM doxycycline (Dox) to induce Cas9 as indicated. Western blot shows FLAG-Cas9 and GAPDH (loading control).


Rag1 - / - : Lig4 - / - : Bcl2 : iCas9 abl pre - B cells were treated with doxycycline as in A and imatinib to trigger cell cycle arrest for 24 h . Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib . All analyses were completed 48 h after gRNA transfection . DSBs quantified by qPCR analysis of Eb ( B ) and Gapdh ( C ) genomic DNA . Results are normalized to cells without gRNA ( - ) . Schematic shows germline ( GL ) locus , unrepaired cut end ( Cut ) and location of gRNA and primers . Loss of germline product is representative of DSB generation .

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

Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with doxycycline as in A and imatinib to trigger cell cycle arrest for 24 h. Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib. All analyses were completed 48 h after gRNA transfection. DSBs quantified by qPCR analysis of Eb (B) and Gapdh (C) genomic DNA. Results are normalized to cells without gRNA (-). Schematic shows germline (GL) locus, unrepaired cut end (Cut) and location of gRNA and primers. Loss of germline product is representative of DSB generation.


( D ) Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified .

O O O O O O O O O O O O O O O O O O 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) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


( E ) Western blot analysis of FLAG - tagged Cas9 or RAG1 ( as indicated ) , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control . Responses to RAG DSBs in Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B cells transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison . # denotes band for FLAG - Cas9 . * denotes band for FLAG - RAG1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of FLAG-tagged Cas9 or RAG1 (as indicated), p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control. Responses to RAG DSBs in Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison. # denotes band for FLAG-Cas9. * denotes band for FLAG-RAG1.


( F ) Cd40 and Pim2 mRNA expression . Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison .

O O O O O 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) Cd40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.


A - B . DSBs quantified by qPCR analysis of Jκ1 ( A ) and JH1 and JHD1 - 1 ( B ) genomic DNA . Schematics show germline ( GL ) locus and location of respective gRNAs and primers . PCR is normalized to cells without gRNA ( - ) . Loss of germline product is representative of DSB generation .

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

A-B. DSBs quantified by qPCR analysis of Jκ1 (A) and JH1 and JHD1-1 (B) genomic DNA. Schematics show germline (GL) locus and location of respective gRNAs and primers. PCR is normalized to cells without gRNA (-). Loss of germline product is representative of DSB generation.


C . Representative images of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows percentage of cells with indicated number of foci from three biological replicates . In each replicate , 100 cells per condition were quantified .

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

C. Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


D . Western blot analysis of FLAG - Cas9 , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control .

O O O O 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. Western blot analysis of FLAG-Cas9, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.


E . CD40 and Pim2 mRNA expression . Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison .

E. CD40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.


B . DSBs quantified by qPCR analysis of Igh ( JH1 ) genomic DNA and analyzed as in Fig 1E with results normalized to empty vector control , which has only germline Igh DNA . There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full - length RAG1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in Fig 1E with results normalized to empty vector control, which has only germline Igh DNA. There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full-length RAG1.


D . Western blot analysis of FLAG - RAG1 , p - KAP1 , NF - κB2 , and PIM2 . GAPDH is shown as loading control .

D. Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.


E. Cd40 and Pim2 mRNA expression.


A . Detection of Sufu and Skp1 after immunoprecipitation of the indicated Flag - tagged F - Box Proteins ( FBPs ) . An Empty Vector ( EV ) was used as a negative control . HEK293T cells were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Detection of Sufu and Skp1 after immunoprecipitation of the indicated Flag-tagged F-Box Proteins (FBPs). An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


B , C . Immunoprecipitation of endogenous Sufu from PC3 ( B ) and DAOY cells ( C ) transfected with either an empty vector ( EV ) or Myc - tagged Fbxl17 . Non - specific rabbit immunoglobulin G ( IgG ) was used as a negative control . Detection of light chains of immunoglobulin ( IgG ) was used to assess the amount of IgG used for each immunoprecipitation reaction . Treatment with MLN4924 ( 2 M ) was started 5 hours before cell collection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoprecipitation of endogenous Sufu from PC3 (B) and DAOY cells (C) transfected with either an empty vector (EV) or Myc-tagged Fbxl17. Non-specific rabbit immunoglobulin G (IgG) was used as a negative control. Detection of light chains of immunoglobulin (IgG) was used to assess the amount of IgG used for each immunoprecipitation reaction. Treatment with MLN4924 (2 M) was started 5 hours before cell collection.


D . Quantification of Fbxl17 mRNA levels in PC3 and DAOY transfected with a non - targeting siRNA ( Control ) or two siRNAs to Fbxl17 ( 1 ) and ( 2 ) . Cells were serum starved for 24 hours in serum - reduced medium and treated with SAG ( 100 nM ) for 24 hours prior collection .

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D. Quantification of Fbxl17 mRNA levels in PC3 and DAOY transfected with a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2). Cells were serum starved for 24 hours in serum-reduced medium and treated with SAG (100 nM) for 24 hours prior collection.


E . Sufu protein levels in PC3 and DAOY cells treated as in D . GAPDH was used as a loading control . Representative image of three independent experiments is shown .

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E. Sufu protein levels in PC3 and DAOY cells treated as in D. GAPDH was used as a loading control. Representative image of three independent experiments is shown.


F . Sufu protein levels in PC3 and DAOY cells infected with an empty backbone retrovirus ( EV ) or a retrovirus expressing Myc - tagged Fbxl17 . Representative image from three independent experiments is shown .

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F. Sufu protein levels in PC3 and DAOY cells infected with an empty backbone retrovirus (EV) or a retrovirus expressing Myc-tagged Fbxl17. Representative image from three independent experiments is shown.


G . Detection of Sufu protein levels following cycloheximide ( CHX ) treatment for the indicated hours , and Fbxl17 depletion using two different siRNAs . ( Hrs = hours ) . Representative image of two independent experiments is

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G. Detection of Sufu protein levels following cycloheximide (CHX) treatment for the indicated hours, and Fbxl17 depletion using two different siRNAs. (Hrs=hours). Representative image of two independent experiments is shown


A . Detection of polyubiquitylated species of endogenous Sufu co - immunopurified from DAOY cells transfected with Myc - tagged ubiquitin ( Ub ) either in the presence of non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) and ( 2 ) . MG132 ( 10µM ) was added in all samples .

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A. Detection of polyubiquitylated species of endogenous Sufu co-immunopurified from DAOY cells transfected with Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.


B . Detection of polyubiquitylated species of Sufu upon co - transfection of HEK293T cells with HA - tagged Sufu and Myc - tagged ubiquitin ( Ub ) either in the presence of non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) and ( 2 ) . MG132 ( 10µM ) was added in all samples .

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B. Detection of polyubiquitylated species of Sufu upon co-transfection of HEK293T cells with HA-tagged Sufu and Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.


C . Fbxl17 mRNA relative levels in DAOY and HEK293T cells upon Fbxl17 depletion with either a non - targeting siRNA ( Control ) or two siRNAs to Fbxl17 ( 1 ) and ( 2 ) .

C. Fbxl17 mRNA relative levels in DAOY and HEK293T cells upon Fbxl17 depletion with either a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2).


D . Detection of ubiquitylated Sufuco - immunoprecipitated from HEK293T cells co - transfected with HA - tagged Sufu , Myc - ubiquitin ( Ub ) along with either Flag - tagged Fbxl17 wild type ( WT ) or a mutant lacking the F - box domain ( Fbxl17ΔF ) . MG132 ( 10µM ) was added in all samples .

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D. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with HA-tagged Sufu, Myc-ubiquitin (Ub) along with either Flag-tagged Fbxl17 wild type (WT) or a mutant lacking the F-box domain (Fbxl17ΔF). MG132 (10µM) was added in all samples.


E . Detection of ubiquitylated Sufuco - immunoprecipitated from HEK293T cells co - transfected with Myc - tagged ubiquitin ( Ub ) , Flag - tagged Fbxl17 and HA - tagged Sufu WT or Sufu mutant K257R . MG132 ( 10µM ) was added in all samples .

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E. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with Myc-tagged ubiquitin (Ub), Flag-tagged Fbxl17 and HA-tagged Sufu WT or Sufu mutant K257R. MG132 (10µM) was added in all samples.


A . Detection of Myc - tagged Fbxl17 after immunoprecipitation of HA - tagged Sufu WT or Sufu S342 / 6A and S342 / 6D , as indicated . HEK293T cells were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

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A. Detection of Myc-tagged Fbxl17 after immunoprecipitation of HA-tagged Sufu WT or Sufu S342/6A and S342/6D, as indicated. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


B . Detection of Flag - tagged Gli1 and Myc - tagged Fbxl17 binding to immunoprecipitated HA - tagged Sufu Wild - Type ( WT ) or to Sufu S352F . An Empty Vector ( EV ) was used as a negative control . HEK293T cells were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

B. Detection of Flag-tagged Gli1 and Myc-tagged Fbxl17 binding to immunoprecipitated HA-tagged Sufu Wild-Type (WT) or to Sufu S352F. An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


C . Detection of phosphorylated Sufu on S352 / T353 after immunoprecipitation of HA - tagged Sufu WT and Sufu 351 - 353 AAA , as indicated .

C. Detection of phosphorylated Sufu on S352/T353 after immunoprecipitation of HA-tagged Sufu WT and Sufu 351-353 AAA, as indicated.


D . Detection of Myc - tagged Fbxl17 binding to Sufu full length ( FL ) or to the following Sufu peptides : Sufu 340 - 360 ( APSRKDSLESDSSTAIIPHEL ) ; Sufu 340 - 360 [ S352P / T353P ] ( phosphorylated on the residues S352 and T353 ) ; Sufu 351 - 372 ( SSTAIIPHELIRTRQLESVHLK ) .

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D. Detection of Myc-tagged Fbxl17 binding to Sufu full length (FL) or to the following Sufu peptides: Sufu 340-360(APSRKDSLESDSSTAIIPHEL); Sufu 340-360 [S352P/T353P] (phosphorylated on the residues S352 and T353); Sufu 351-372 (SSTAIIPHELIRTRQLESVHLK).


E . Detection of HA - tagged Sufu binding to a Myc - tagged Fbxl17 construct encompassing the residues 318 to 701 ( corresponding to the F - box motif and C - terminus region ) assessed by in vitro binding assay . Both proteins were synthetized in vitro using a T7 - coupled reticulocyte lysate system .

E. Detection of HA-tagged Sufu binding to a Myc-tagged Fbxl17 construct encompassing the residues 318 to 701 (corresponding to the F-box motif and C-terminus region) assessed by in vitro binding assay. Both proteins were synthetized in vitro using a T7-coupled reticulocyte lysate system.


F . Detection of ubiquitylated Sufu immunoprecipitated from HEK293T cells co - transfected with HA - tagged ubiquitin ( Ub ) , Myc - tagged Fbxl17 and Flag - tagged Gli1 . MG132 ( 10µM ) was added in all samples .

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F. Detection of ubiquitylated Sufu immunoprecipitated from HEK293T cells co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged Fbxl17 and Flag-tagged Gli1. MG132 (10µM) was added in all samples.


G . Detection of Flag - tagged Gli1 binding to Myc - tagged Fbxl17 co - immunoprecipitated from HEK293T cells transfected with a non - targeting siRNA ( Control ) or two siRNA to Sufu ( 1 ) and ( 2 ) , as indicated .

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G. Detection of Flag-tagged Gli1 binding to Myc-tagged Fbxl17 co-immunoprecipitated from HEK293T cells transfected with a non-targeting siRNA (Control) or two siRNA to Sufu (1) and (2), as indicated.


A . Sufu protein levels in mouse embryonic fibroblasts ( MEFs ) Ptch1 + / + , Ptch1 + / - and Ptch1 - / - transfected with a non - targeting siRNA ( Control ) or two siRNAs targeting mouse Fbxl17 ( 1 ) and ( 2 ) .

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A. Sufu protein levels in mouse embryonic fibroblasts (MEFs) Ptch1+/+, Ptch1+/- and Ptch1-/- transfected with a non-targeting siRNA (Control) or two siRNAs targeting mouse Fbxl17 (1) and (2).


B . Quantification of Fbxl17 mRNA levels in MEFs Ptch1 + / + , Ptch1 + / - and Ptch1 - / - transfected as in A .

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B. Quantification of Fbxl17 mRNA levels in MEFs Ptch1+/+, Ptch1+/- and Ptch1-/- transfected as in A.


C . Analysis of Gli1 mRNA levels in MEFs Ptch1 + / + and Ptch1 - / - upon Fbxl17 depletion using two different siRNAs . ( mean ± SEM from 3 independent experiments , * * * p < 0 . 0005 ) .

C. Analysis of Gli1 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, ***p<0.0005).


D . Analysis of Bcl2 mRNA levels in MEFs Ptch1 + / + and Ptch1 - / - upon Fbxl17 depletion using two different siRNAs . ( mean ± SEM from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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D. Analysis of Bcl2 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


E . Detection of Flag - tagged Gli1 binding to HA - tagged Sufu in MEFs Ptch1 - / - cells upon expression of Myc - tagged Fbxl17 , or Fbxl17 depletion by two siRNAs . For each condition , 200 µg of whole cell lysate was immunoprecipitated .

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E. Detection of Flag-tagged Gli1 binding to HA-tagged Sufu in MEFs Ptch1-/- cells upon expression of Myc-tagged Fbxl17, or Fbxl17 depletion by two siRNAs. For each condition, 200 µg of whole cell lysate was immunoprecipitated.


F . Immunostaining of Fbxl17 using anti - Myc antibody in Ptch1 - / - MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at the N - terminus [ Myc ( N ) - Fbxl17 ] or at the C - terminus [ Myc ( C ) - Fbxl17 ] . Scale bars : 20 m .

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F. Immunostaining of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at the N-terminus [Myc(N)-Fbxl17] or at the C-terminus [Myc(C)-Fbxl17]. Scale bars: 20 m.


G . Detection of Fbxl17 using anti - Myc antibody in Ptch1 - / - MEFs stably expressing Myc ( N ) - Fbxl17 ( Fbxl17 tagged at the N - terminus ) and Myc ( C ) - Fbxl17 ( Fbxl17 tagged at the C - terminus ) .

G. Detection of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably expressing Myc(N)-Fbxl17 (Fbxl17 tagged at the N-terminus) and Myc(C)-Fbxl17 (Fbxl17 tagged at the C-terminus).


H . Detection of HA - tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from cytoplasmic and nuclear extracts from Ptch1 - / - MEFs . Asterisk ( * ) indicates a non specific band . Identification of cytoplasmic and nuclear fractions was performed by Lamin A / C and GAPDH detection .

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H. Detection of HA-tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from cytoplasmic and nuclear extracts from Ptch1-/- MEFs. Asterisk (*) indicates a non specific band. Identification of cytoplasmic and nuclear fractions was performed by Lamin A/C and GAPDH detection.


A . Cell proliferation of DAOY cells upon Fbxl17 depletion using a non - targeting siRNA ( Control ) , two siRNAs against Fbxl17 ( 1 ) and ( 2 ) or upon reintroduction of a Myc - tagged Fbxl17 construct in Fbxl17 - depleted DAOY cells using siRNA Fbxl17 ( 2 ) ( mean ± SEM from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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A. Cell proliferation of DAOY cells upon Fbxl17 depletion using a non-targeting siRNA (Control), two siRNAs against Fbxl17 (1) and (2) or upon reintroduction of a Myc-tagged Fbxl17 construct in Fbxl17-depleted DAOY cells using siRNA Fbxl17 (2) (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


B . Quantification of Fbxl17 mRNA levels in DAOY cells transfected with a non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) and ( 2 ) ( mean ± SEM from 3 independent experiments , * * p < 0 . 005 ) .

B. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with a non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2) (mean ± SEM from 3 independent experiments, **p<0.005).


C . Sufu protein levels in DAOY cells treated as in A . Representative image of three independent experiments is shown .

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C. Sufu protein levels in DAOY cells treated as in A. Representative image of three independent experiments is shown.


D . Cell proliferation of DAOY cells transfected with non - targeting siRNA ( Control ) , siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 and Sufu . ( mean ± SEM from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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D. Cell proliferation of DAOY cells transfected with non-targeting siRNA (Control), siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 and Sufu. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


E . Quantification of Fbxl17 mRNA levels in DAOY cells treated as in D mean ± SEM from 3 independent experiments , * * * p < 0 . 0005 ) .

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E. Quantification of Fbxl17 mRNA levels in DAOY cells treated as in D mean ± SEM from 3 independent experiments, ***p<0.0005).


F . Sufu protein levels in DAOY cells treated as in D . Representative image of three independent experiments is shown .

F. Sufu protein levels in DAOY cells treated as in D. Representative image of three independent experiments is shown.


A . Quantification of Fbxl17 mRNA levels in DAOY cells transfected with control shRNA or shRNA targeting Fbxl17 . ( mean ± SEM from 3 independent experiments , * * * p < 0 . 0005 ) .

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A. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with control shRNA or shRNA targeting Fbxl17. (mean ± SEM from 3 independent experiments, ***p<0.0005).


B . Detection of Sufu protein levels in DAOY cells transfected with control shRNA or shRNA Fbxl17 . Representative image of three independent experiments is shown .

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B. Detection of Sufu protein levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. Representative image of three independent experiments is shown.


C . Quantification of Gli1 mRNA levels in DAOY cells transfected with control shRNA or shRNA Fbxl17 . ( mean ± SEM from 3 independent experiments , * * p < 0 . 005 ) .

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C. Quantification of Gli1 mRNA levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. (mean ± SEM from 3 independent experiments, **p<0.005).


D . T2 - weighted magnetic resonance imaging ( MRI ) showing tumour development in rats injected with DAOY cells transfected with control shRNA or shRNA against Fbxl17 . One representative image for each condition , at 10 weeks is shown . Scale bar : 5mm . E . Graph showing area of T2 hyperintensity ( a surrogate marker of tumourgrowth ) between 4 and 10 weeks post - tumour induction . Animals were injected with DAOY cells stably expressing control shRNA or shRNA against Fbxl17 ( 10 , 000 cells / 1µL ) . ( mean ± SEM ; n = 5 ; two - way ANOVA , followed by unpaired t - test , * p < 0 . 05 , * * p < 0 . 01 ) .

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D. T2- weighted magnetic resonance imaging (MRI) showing tumour development in rats injected with DAOY cells transfected with control shRNA or shRNA against Fbxl17. One representative image for each condition, at 10 weeks is shown. Scale bar: 5mm. E. Graph showing area of T2 hyperintensity (a surrogate marker of tumourgrowth) between 4 and 10 weeks post-tumour induction. Animals were injected with DAOY cells stably expressing control shRNA or shRNA against Fbxl17 (10,000 cells/1µL). (mean ± SEM; n = 5; two-way ANOVA, followed by unpaired t-test, *p<0.05, **p<0.01).


F . Representative immunohistochemistry image of Vimentin staining . Scale bar : 1 mm . G . Quantification of tumour growth ( vimentin staining ) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 ( mean ± SEM ; n = 5 ; unpaired t - test , * p < 0 . 05 ) .

F. Representative immunohistochemistry image of Vimentin staining. Scale bar: 1 mm.G. Quantification of tumour growth (vimentin staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).


H . Representative immunohistochemistry image of Ki67 staining . Scale bar : 0 . 5 mm / 0 . 1 mm . I . Quantification of proliferative index ( Ki67 staining ) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 ( mean ± SEM ; n = 5 ; unpaired t - test , * p < 0 . 05 ) .

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H. Representative immunohistochemistry image of Ki67 staining. Scale bar: 0.5 mm/0.1 mm.I. Quantification of proliferative index (Ki67 staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).


A - C . mRNA expression of Sufu , Gli1 and Fbxl17 detected in 285 medulloblastoma samples ( GEO accession : GSE37382 ) , stratified by tumor subtype . Level of significance ( p ) for Kruskal - Wallis ( KW ) rank test is p < 10 - 8 . Analysis using one - way anova gave comparable results .

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A-C. mRNA expression of Sufu, Gli1 and Fbxl17 detected in 285 medulloblastoma samples (GEO accession: GSE37382), stratified by tumor subtype. Level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8. Analysis using one-way anova gave comparable results.