Figure 2 ( A ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot . ( B ) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot . ( C ) Macrophages were pre - treated with either 1 μM GSK inh or 0 . 1 μM MLi - 2 and then treated with 1 mM LLOMe for 30 min . Rab8A and Rab8A pT72 levels were analysed by Western blot . ( D - F ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ( D ) Endogenous LRRK2 and ( E ) Rab8A recruitment to LAMP - 1 positive compartments was visualised by immunofluorescence . Scale bar = 10 μm . ( F ) Quantification of D - E . Data represents the mean ± SEM of three independent biological experiments . LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min . The number of LRRK2 per cell were monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . Data represent the mean ± SEM of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s test against the untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 . LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min . The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . Data represent the mean ± SEM of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s test against the untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 .

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Figure 2(A) RAW264.7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot.(B) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot.(C) Macrophages were pre-treated with either 1 μM GSK inh or 0.1 μM MLi-2 and then treated with 1 mM LLOMe for 30 min. Rab8A and Rab8A pT72 levels were analysed by Western blot.(D-F) RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (D) Endogenous LRRK2 and (E) Rab8A recruitment to LAMP-1 positive compartments was visualised by immunofluorescence. Scale bar = 10 μm. (F) Quantification of D-E. Data represents the mean ± SEM of three independent biological experiments.LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of LRRK2 per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.


Figure 3 ( A ) RAW264 . 7 macrophages were electroporated with EGFP - Rab8A and treated with 1 mM LLOMe for 30 min . Intracellular distribution of Galectin - 3 , CHMP4B or LC3B was visualised by immunofluorescence . Scale bar = 5 μm . ( B ) Macrophages were electroporated with EGFP - Rab8A and RFP - Galectin - 3 . Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging . Snapshots at the indicated time points after LLOMe addition are shown . Scale bar = 5 μm . ( C ) RAW264 . 7 macrophages were treated with 1 mM of LLOMe and LRRK2 , Rab8A , CHMP4B , Galectin - 3 and LC3B foci over time was analysed by high - content imaging . Data shows the mean ± SEM of five biological replicates . RAW264 . 7 macrophages were pre - treated with 10 μM BAPTA - AM for 1 hr , and treated with 1 mM LLOMe for 30 min . ( D ) CHMP4B recruitment was monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . ( E ) Rab8A pT72 phosphorylation was analysed by Western blot . ( F ) LRRK2 and ( G ) Rab8A recruitment was monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . ( H ) Quantification of E and F .

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Figure 3(A) RAW264.7 macrophages were electroporated with EGFP-Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin-3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm.(B) Macrophages were electroporated with EGFP-Rab8A and RFP-Galectin-3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm.(C) RAW264.7 macrophages were treated with 1 mM of LLOMe and LRRK2, Rab8A, CHMP4B, Galectin-3 and LC3B foci over time was analysed by high-content imaging. Data shows the mean ± SEM of five biological replicates.RAW264.7 macrophages were pre-treated with 10 μM BAPTA-AM for 1 hr, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm.(E) Rab8A pT72 phosphorylation was analysed by Western blot.(F) LRRK2 and (G) Rab8A recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (H) Quantification of E and F.


Figure 4RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show the quantification of number of CHMP4B positive vesicles per cell . Mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test . RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . Galectin - 3 vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show the quantification of number of Galectin - 3 positive vesicles per cell . Mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test . Live cell imaging of LysoTracker positive spots in WT , LRRK2 KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in the LLOMe treatment window were estimated using linear regression . Live cell imaging of LysoTracker positive spots in WT , Rab8A KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in the LLOMe treatment window were estimated using linear regression .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. Galectin-3 vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of Galectin-3 positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.Live cell imaging of LysoTracker positive spots in WT, LRRK2 KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.Live cell imaging of LysoTracker positive spots in WT, Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.


Figure 5WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) and WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show quantification of number of positive vesicles per cell . Data show the mean ± SEM of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test . WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) and WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show quantification of number of positive vesicles per cell . Data show the mean ± SEM of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test . ( C ) Representative images of LC3B / LAMP - 1 double positive endolysosomes in control and LLOMe treated macrophages . Scale bar = 10 μm . ( D ) Analysis of the % of LC3B / LAMP - 1 double positive endolysosomes in LLOMe treated macrophages . Data shows mean ± SEM of three independent biological experiments . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 by Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.(C) Representative images of LC3B/LAMP-1 double positive endolysosomes in control and LLOMe treated macrophages. Scale bar = 10 μm. (D) Analysis of the % of LC3B/LAMP-1 double positive endolysosomes in LLOMe treated macrophages. Data shows mean ± SEM of three independent biological experiments. *p≤0.05, ***p≤0.001 by Student's t-test.


Figure 6WT and LRRK2 KO RAW264 . 7 macrophages were infected with Mtb for 24 h , Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test . WT and LRRK2 KO RAW264 . 7 macrophages were infected with Ca for 60 min Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test . WT and LRRK2 KO RAW264 . 7 macrophages were infected with Lm for 60 min . Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test . WT and LRRK2 KO RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test . WT and LRRK2 KO RAW264 . 7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h . Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test . WT and LRRK2 KO RAW264 . 7 macrophages were infected with Lm WT or Lm Δhly . Growth measured for 2 h . Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb for 24 h, Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.WT and LRRK2 KO RAW264.7 macrophages were infected with Ca for 60 min Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.WT and LRRK2 KO RAW264.7 macrophages were infected with Lm for 60 min. Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.WT and LRRK2 KO RAW264.7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.WT and LRRK2 KO RAW264.7 macrophages were infected with Lm WT or Lm Δhly. Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


Figure 7Monocyte - derived macrophages from healthy controls and PD patients carrying the G2019S ( Donor 2 - 3 ) or R1441C ( Donor 1 ) LRRK2 mutation were pre - treated with 0 . 1 μM MLi - 2 and treated with 1 mM LLOMe for 30 min . ( A ) Rab8A and Rab8A pT72 levels were analysed by Western blot . ( B ) Rab8A and Galectin - 3 positive vesicle numbers were analysed by immunofluorescence . Data show the mean ± SEM of biological replicates . Scale bar = 20 μm . ns = non - significant , * * p ≤ 0 . 01 , by One - way ANOVA followed by Sidak ' s multiple comparisons test .

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Figure 7Monocyte-derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2-3) or R1441C (Donor 1) LRRK2 mutation were pre-treated with 0.1 μM MLi-2 and treated with 1 mM LLOMe for 30 min. (A) Rab8A and Rab8A pT72 levels were analysed by Western blot. (B) Rab8A and Galectin-3 positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns=non-significant, **p≤0.01, by One-way ANOVA followed by Sidak's multiple comparisons test.


Figure 1a , Survival curves of wild type ( n = 27 ) and Cdc14 DKO ( n = 51 ) mice during the first 6 weeks of life . ( * * * , p < 0 . 001 ; Log - rank , Mantel - Cox test ) . b , Representative images of postnatal day 9 Cdc14 DKO and wild - type littermates . c , Weight of Cdc14 DKO and wild - type littermates during the first 3 weeks of life ( n = 4 per genotype ) . Data information : Bars indicate mean ± SEM . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . d , Representative macroscopic pictures of brain dorsal view of wild type ( WT ) and Cdc14 KO mice at postnatal 0 , 5 and 9 days ( P0 , P5 and P9 ) . Scale bar , 2 mm . The bar plots show the quantification of brain width , length and area , respectively . Data information : Bars indicate mean ± SEM . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice . e , Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0 , P5 and P9 . Fissures underdeveloped in mutant samples are labeled with arrowheads . Scale bar , 700 μm . The bar plots show the quantification of the cerebellar vermis area , the number of folia and the average length of the fissures , respectively . Data information : Bars indicate mean ± SEM . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice . f , Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples . The molecular layer ( ML ) , internal granule cell layer ( IGL ) and external granule cell layer ( EGL ) are indicated . The width of these layers is shown in the bar plots . Data information : Bars indicate mean ± SEM . * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice . In f , each point represents the average value of 9 different random measurements across distinctive folia for each mouse . g , Representative images of misdigital sections stained with DAPI ( blue ) , Calbindin ( green ) and GFAP ( red ) of cerebellar vermis of wild - type ad Cdc14 DKO mice at 9 days after birth . Scale bar , 50 μm . Plots show the quantification of Purkinje cell dendritic length ( average distance of 30 random measurements ) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot . Data information : Bars indicate mean ± SEM . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1a, Survival curves of wild type (n=27) and Cdc14 DKO (n=51) mice during the first 6 weeks of life. (***, p<0.001; Log-rank, Mantel-Cox test). b, Representative images of postnatal day 9 Cdc14 DKO and wild-type littermates. c, Weight of Cdc14 DKO and wild-type littermates during the first 3 weeks of life (n=4 per genotype). Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.d, Representative macroscopic pictures of brain dorsal view of wild type (WT) and Cdc14 KO mice at postnatal 0, 5 and 9 days (P0, P5 and P9). Scale bar, 2 mm. The bar plots show the quantification of brain width, length and area, respectively. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.e, Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0, P5 and P9. Fissures underdeveloped in mutant samples are labeled with arrowheads. Scale bar, 700 μm. The bar plots show the quantification of the cerebellar vermis area, the number of folia and the average length of the fissures, respectively. Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.f, Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples. The molecular layer (ML), internal granule cell layer (IGL) and external granule cell layer (EGL) are indicated. The width of these layers is shown in the bar plots. Data information: Bars indicate mean ± SEM. *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice. In f, each point represents the average value of 9 different random measurements across distinctive folia for each mouse.g, Representative images of misdigital sections stained with DAPI (blue), Calbindin (green) and GFAP (red) of cerebellar vermis of wild-type ad Cdc14 DKO mice at 9 days after birth. Scale bar, 50 μm. Plots show the quantification of Purkinje cell dendritic length (average distance of 30 random measurements) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.


Figure 2a . Confocal microscopy analysis of wild - type and Cdc14 DKO embryonic stem cells ( ESCs ) subjected to neural differentiation . Neuronal marker TUJ1 ( green ) , glial marker GFAP ( red ) and nuclear marker DAPI ( blue ) are stained and quantified after 18 days in differentiation media . Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones . * * * * p - value < 0 . 0001 . Student ' s t - test . Scale bar 50 μm . c , Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation . Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media . Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells . * * * * , p < 0 . 0001 ; Students ' t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2a. Confocal microscopy analysis of wild-type and Cdc14 DKO embryonic stem cells (ESCs) subjected to neural differentiation. Neuronal marker TUJ1 (green), glial marker GFAP (red) and nuclear marker DAPI (blue) are stained and quantified after 18 days in differentiation media. Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones. **** p-value<0.0001. Student's t-test. Scale bar 50 μm.c, Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation. Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media. Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells. ****, p<0.0001; Students' t-test.


Figure 4d , Western blot analysis of CDC14B - GFP co - immunoprecipitations . HeLa cells were transfected with UTF1 and GFP or CDC14B - GFP . Cytoplasmic and nuclear soluble proteins were extracted with RIPA , and nucleolar and chromatin - bound proteins were extracted with high salt RIPA . Immunoprecipitations with anti - GFP antibody were performed . Total protein extracts and immunoprecipitates were loaded and UTF ( upper panel ) and GFP ( lower panel ) were blotted . e , Immunoprecipitation of UTF1 upon co - transfection with CDC14B . Total protein was extracted with high - salt RIPA and immunoprecipitations with anti - UTF1 antibody were performed . f , Expression of active CDC14B , but not a phosphatase - dead ( PD ) isoform , reduces slow - mobility bands of UTF1 . Vinculin was used as a loading control . h , In vitro kinase assay of recombinant human UTF1 with ERK2 , CDK1 - Cyclin B1 , CDK1 - Cyclin A2 or in absence of kinases ( C ) . j , In vitro phosphatase assay of UTF1 . Three different fragments of UTF1 were phosphorylated by CDK1 - Cyclin A2 and subsequently exposed to wild - type or a catalytically inactive form ( PD ) of CDC14B .

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Figure 4d, Western blot analysis of CDC14B-GFP co-immunoprecipitations. HeLa cells were transfected with UTF1 and GFP or CDC14B-GFP. Cytoplasmic and nuclear soluble proteins were extracted with RIPA, and nucleolar and chromatin-bound proteins were extracted with high salt RIPA. Immunoprecipitations with anti-GFP antibody were performed. Total protein extracts and immunoprecipitates were loaded and UTF (upper panel) and GFP (lower panel) were blotted.e, Immunoprecipitation of UTF1 upon co-transfection with CDC14B. Total protein was extracted with high-salt RIPA and immunoprecipitations with anti-UTF1 antibody were performed.f, Expression of active CDC14B, but not a phosphatase-dead (PD) isoform, reduces slow-mobility bands of UTF1. Vinculin was used as a loading control.h, In vitro kinase assay of recombinant human UTF1 with ERK2, CDK1-Cyclin B1, CDK1-Cyclin A2 or in absence of kinases (C).j, In vitro phosphatase assay of UTF1. Three different fragments of UTF1 were phosphorylated by CDK1-Cyclin A2 and subsequently exposed to wild-type or a catalytically inactive form (PD) of CDC14B.


Figure 5a , Quantitative RT - PCR in wild - type ( WT ) or Cdc14 DKO ESCs of the indicated transcripts during neural differentiation . n = 3 independent experiments ( ± S . E . M . ) ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Student ' s t - test . b , Expression of a CDC14 - GFP fusion protein in ESCs before ( T0 ) and after three days ( T3 ) in differentiation conditions . The arrowheads indicate the concentration of CDC14 - GFP in the nucleolus before but not after the induction of differentiation . Scale bars , 5 μM . The plot shows the ratio between nucleolar vs . total nuclear URF1 intensity at T0 ( n = 26 cells ) or T3 ( n = 31 ) in cells from two separate experiments . * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . c , Confocal microscopy analysis of UTF1 intensity in WT and Cdc14 DKO ESCs at time 0 ( T0 ) and after 1 day ( T1 ) in differentiation media . The quantification of UTF1 fluorescence mean intensity ( FMI ) per cell is shown in the plot to the right . Figures and horizontal bars indicate mean ± S . E . M . ( n = 1269 WT T0 cells , n = 2726 WT T1 , n = 974 Cdc14 DKO T0 , n = 1173 Cdc14 DKO T1 ) . * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; Student ' s t - test with Welch ' s correction . d , Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs at time 0 and after 3 days in differentiation media . e , Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs in absence or presence of the kinase inhibitors roscovitine ( R ) or trametinib ( T ) . f , Immunoblot analysis of UTF1 protein stability after transfection of HeLa cells with UTF1 and GFP or CDC14B - GFP . Cells were treated with cycloheximide ( CHX ) and total proteins were detected at different time points as indicated . Cyclin B1 and β - catenin are used as controls . g , Western blot analysis of UTF1 WT or mutants in three ( UTF1 - 3KR ) or five ( UTF1 - 5KR ) lysines in absence or presence of trametinib ( T ) or roscovitine ( R ) . h , Immunoblot of UTF1 mutants in SPOP , SIAH or SPOP + SIAH potential binding sites , as well as S48D and S54D phosphorylation sites . Actin was used as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5a, Quantitative RT-PCR in wild-type (WT) or Cdc14 DKO ESCs of the indicated transcripts during neural differentiation. n=3 independent experiments (±S.E.M.); *, p<0.05; **, p<0.01; ***, p<0.001; Student's t-test.b, Expression of a CDC14-GFP fusion protein in ESCs before (T0) and after three days (T3) in differentiation conditions. The arrowheads indicate the concentration of CDC14-GFP in the nucleolus before but not after the induction of differentiation. Scale bars, 5 μM. The plot shows the ratio between nucleolar vs. total nuclear URF1 intensity at T0 (n= 26 cells) or T3 (n=31) in cells from two separate experiments. ***, p<0.001, Student's t-test with Welch's correction.c, Confocal microscopy analysis of UTF1 intensity in WT and Cdc14 DKO ESCs at time 0 (T0) and after 1 day (T1) in differentiation media. The quantification of UTF1 fluorescence mean intensity (FMI) per cell is shown in the plot to the right. Figures and horizontal bars indicate mean ± S.E.M. (n=1269 WT T0 cells, n=2726 WT T1, n=974 Cdc14 DKO T0, n=1173 Cdc14 DKO T1). **, p<0.01; ****, p<0.0001; Student's t-test with Welch's correction.d, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs at time 0 and after 3 days in differentiation media.e, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs in absence or presence of the kinase inhibitors roscovitine (R) or trametinib (T).f, Immunoblot analysis of UTF1 protein stability after transfection of HeLa cells with UTF1 and GFP or CDC14B-GFP. Cells were treated with cycloheximide (CHX) and total proteins were detected at different time points as indicated. Cyclin B1 and β-catenin are used as controls.g, Western blot analysis of UTF1 WT or mutants in three (UTF1-3KR) or five (UTF1-5KR) lysines in absence or presence of trametinib (T) or roscovitine (R).h, Immunoblot of UTF1 mutants in SPOP, SIAH or SPOP+SIAH potential binding sites, as well as S48D and S54D phosphorylation sites. Actin was used as a loading control.


Figure 1 ( A ) Representative whole mount images of WT and Cep63T / T brains at P60 . Scale bar = 0 . 2 cm . ( B ) Telencephalon area of WT and Cep63T / T brains . WT littermates N = 4 , Cep63T / T N = 7 ; two - tailed Mann - Whitney t - test . ( C ) Representative whole mount images of WT and Sas4cKO brains at P14 . Scale bar : 0 . 2 cm . ( D ) Telencephalon area of WT and Sas4cKO brains . WT littermates N = 8 , Sas4cKO N = 7 ; two - tailed Mann - Whitney t - test . Black circles represent Nestin - Cre + animals . ( E ) Representative images of E14 . 5 WT , Cep63T / T and Sas4cKO cortices stained with antibodies against α - Tubulin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . Magenta arrows indicate dead cells ; white and yellow arrows indicate cells with bipolar and monopolar spindles , respectively . Insets showing zoomed in view of 2 representative cells . Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface . Scale bar = 25 μm . ( F ) Graph showing the percentage of bipolar , monopolar and multipolar cells at the ventricular surface of E14 . 5 cortices . WT n = 201 cells , N = 3 embryos ; Cep63T / T n = 312 cells , N = 3 embryos ; Sas4cKO n = 296 cells , N = 3 embryos ; # , chi - square test with * , post - hoc analysis , comparisons are made to WT . ( G ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) and phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of cells in the ventricular zone ( VZ ) and sub - ventricular zone ( SVZ ) . Scale bar = 100 μm . ( H ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Sas4cKO N = 3 ; two - tailed Mann - Whitney t - test . ( J ) Representative images from live - cell movies of Nuclear - mCherry + WT , Cep63T / T and Sas4cKO NPCs . The timing of mitosis began at nuclear envelope breakdown ( NEBD ) and finished at nuclear envelope reformation . White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis . ( K ) Plot showing the duration of mitosis in WT , Cep63T / T and Sas4cKO NPCs . Triangles represent individual cells ; triangles of the same color represent cells derived from the same embryo . Circles represent the average mitotic duration of cells from each embryo . WT n = 202 cells , N = 4 embryos ; Cep63T / T n = 141 cells , N = 3 embryos ; Sas4cKO n = 138 cells , N = 3 embryos ; two - tailed Welch ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Representative whole mount images of WT and Cep63T/T brains at P60. Scale bar = 0.2 cm. (B) Telencephalon area of WT and Cep63T/T brains. WT littermates N = 4, Cep63T/T N = 7; two-tailed Mann-Whitney t-test.(C) Representative whole mount images of WT and Sas4cKO brains at P14. Scale bar: 0.2 cm. (D) Telencephalon area of WT and Sas4cKO brains. WT littermates N = 8, Sas4cKO N = 7; two-tailed Mann-Whitney t-test. Black circles represent Nestin-Cre+ animals.(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm. (F) Graph showing the percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T n = 312 cells, N = 3 embryos; Sas4cKO n = 296 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT.(G) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the ventricular zone (VZ) and sub-ventricular zone (SVZ). Scale bar = 100 μm.(H) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T N = 3, Sas4cKO N = 3; two-tailed Mann-Whitney t-test.(J) Representative images from live-cell movies of Nuclear-mCherry+ WT, Cep63T/T and Sas4cKO NPCs. The timing of mitosis began at nuclear envelope breakdown (NEBD) and finished at nuclear envelope reformation. White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis. (K) Plot showing the duration of mitosis in WT, Cep63T/T and Sas4cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Cep63T/T n = 141 cells, N = 3 embryos; Sas4cKO n = 138 cells, N = 3 embryos; two-tailed Welch's t-test.


Figure 2 ( A - C ) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 mins . WT n = 330 cells , N = 4 embryos ; Cep63T / T n = 260 cells , N = 3 embryos ; Sas4cKO n = 223 cells , N = 3 embryos . ( D - F ) Percentage of proliferating , arrested and apoptotic progeny within each group of the indicated mitotic duration , calculated from data shown in A - C ; groups with n < 20 cells were excluded from this quantification ; # , chi - square test with * , post - hoc analysis , within each genotype , comparisons are made to the 0 - 30min group . ( G ) Genome - wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14 . 5 embryos . Individual cells are represented in rows with copy number states indicated by colors ; arrows on the zoomed in view ( right ) indicate aneuploid cells . WT n = 38 cells , N = 2 embryos ; Cep63T / T n = 40 cells , N = 2 embryos ; Sas4cKO n = 42 cells , N = 2 embryos . ( H ) Percentage of aneuploid cells from the single - cell sequencing data shown in G .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-C) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 mins. WT n = 330 cells, N = 4 embryos; Cep63T/T n = 260 cells, N = 3 embryos; Sas4cKO n = 223 cells, N = 3 embryos. (D-F) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in A-C; groups with n < 20 cells were excluded from this quantification; #, chi-square test with *, post-hoc analysis, within each genotype, comparisons are made to the 0-30min group.(G) Genome-wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14.5 embryos. Individual cells are represented in rows with copy number states indicated by colors; arrows on the zoomed in view (right) indicate aneuploid cells. WT n = 38 cells, N = 2 embryos; Cep63T/T n = 40 cells, N = 2 embryos; Sas4cKO n = 42 cells, N = 2 embryos. (H) Percentage of aneuploid cells from the single-cell sequencing data shown in G.


Figure 3 ( A ) Representative whole mount images of WT , Sas4cKO , Sas4cKO ; Trp53bp1 - / - , Sas4cKO ; Usp28cKO animals at P14 . Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia . Scale bar = 0 . 2 cm . ( B ) Telencephalon area of P14 brains of the indicated genotypes . WT littermates N = 8 , Sas4cKO N = 7 , Sas4cKO ; Trp53bp1 - / - N = 6 , Sas4cKO ; Usp28cKO N = 10 , Sas4cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc analysis . WT and Sas4cKO data are from Figure 1D and shown alongside for comparison . Black circles represent Nestin - Cre + animals . ( C ) Cortical thickness of P14 brains of the indicated genotypes . WT littermates N = 4 , Sas4cKO N = 4 , Sas4cKO ; Trp53bp1 - / - N = 4 , Sas4cKO ; Usp28cKO N = 4 , Sas4cKO ; Trp53 - / - N = 4 ; one - way ANOVA with post - hoc analysis . WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison . Black circles represent Nestin - Cre + animals . ( D ) Representative whole mount images of WT , Cep63T / T , Cep63T / T ; Trp53bp1 - / - , Cep63T / T ; Usp28 - / - animals at P60 . Scale bar = 0 . 2 cm . ( E ) Telencephalon area of P60 brains of the indicated genotypes . WT littermates N = 4 , Cep63T / T N = 7 , Cep63T / T ; Trp53bp1 - / - N = 6 , Cep63T / T ; Usp28 - / - N = 5 ; one - way ANOVA with post - hoc analysis . WT and Cep63T / T data are from Figure 1B and shown alongside for comparison . ( F ) Cortical thickness of P60 brains of the indicated genotypes . WT littermates N = 4 , Cep63T / T N = 5 , Cep63T / T ; Trp53bp1 - / - N = 3 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis . WT and Cep63T / T data are from Figure EV1B and shown alongside for comparison . ( G ) Body weight of P7 animals of the indicated genotypes . WT littermates N = 18 , Cep63T / T N = 16 , Cep63T / T ; Trp53bp1 - / - N = 2 , Cep63T / T ; Usp28 - / - N = 10 ; one - way ANOVA with post - hoc analysis . ( H ) Quantification of total number of cells within a 500 μm - width column of cortices at P60 . WT littermates N = 4 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis . ( I - J ) Quantification of the number of cells in the ( I ) superficial layer ( CUX1 + ) and ( J ) deep layer ( CTIP2 + ) within a 500 μm - width column of P60 cortices . WT littermates N = 3 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc analysis . ( K ) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 ( green ) , superficial layer marker CUX1 ( red ) and DAPI ( blue ) . Scale bar = 200 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative whole mount images of WT, Sas4cKO, Sas4cKO;Trp53bp1-/-, Sas4cKO;Usp28cKO animals at P14. Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia. Scale bar = 0.2 cm. (B) Telencephalon area of P14 brains of the indicated genotypes. WT littermates N = 8, Sas4cKO N = 7, Sas4cKO;Trp53bp1-/- N = 6, Sas4cKO;Usp28cKO N = 10, Sas4cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure 1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals. (C) Cortical thickness of P14 brains of the indicated genotypes. WT littermates N = 4, Sas4cKO N = 4, Sas4cKO;Trp53bp1-/- N = 4, Sas4cKO;Usp28cKO N = 4, Sas4cKO;Trp53-/- N = 4; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals.(D) Representative whole mount images of WT, Cep63T/T, Cep63T/T;Trp53bp1-/-, Cep63T/T ;Usp28-/- animals at P60. Scale bar = 0.2 cm. (E) Telencephalon area of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 7, Cep63T/T;Trp53bp1-/- N = 6, Cep63T/T;Usp28-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure 1B and shown alongside for comparison. (F) Cortical thickness of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 5, Cep63T/T;Trp53bp1-/- N = 3, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure EV1B and shown alongside for comparison.(G) Body weight of P7 animals of the indicated genotypes. WT littermates N=18, Cep63T/T N=16, Cep63T/T;Trp53bp1-/- N=2, Cep63T/T;Usp28-/- N=10; one-way ANOVA with post-hoc analysis.(H) Quantification of total number of cells within a 500 μm-width column of cortices at P60. WT littermates N = 4, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .(I-J) Quantification of the number of cells in the (I) superficial layer (CUX1+) and (J) deep layer (CTIP2+) within a 500 μm-width column of P60 cortices. WT littermates N = 3, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .(K) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 (green), superficial layer marker CUX1 (red) and DAPI (blue). Scale bar = 200 μm.


Figure 4 ( A ) Cortices from E14 . 5 brains stained with antibodies against the radial glial cell marker PAX6 ( yellow ) , intermediate progenitor marker TBR2 ( red ) and DAPI ( blue ) . Scale bar = 50 μm . ( B and C ) Quantification of the number of ( B ) radial glial cells ( PAX6 + ) and ( C ) intermediate progenitors ( TBR2 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 3 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 3 , Sas4cKO ; Usp28cKO N = 3 ; one - way ANOVA with post - hoc analysis . ( D ) Cortices from E14 . 5 embryos stained with antibodies against Cleaved - Caspase 3 ( CC3 , white ) , TP53 ( red ) and DAPI . Scale bar = 50 μm . ( E and F ) Quantification of the number of CC3 + apoptotic cells ( E ) and cells with TP53 activation ( F ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 4 , Sas4cKO ; Usp28cKO N = 4 ; one - way ANOVA with post - hoc analysis .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Cortices from E14.5 brains stained with antibodies against the radial glial cell marker PAX6 (yellow), intermediate progenitor marker TBR2 (red) and DAPI (blue). Scale bar = 50 μm. (B and C) Quantification of the number of (B) radial glial cells (PAX6+) and (C) intermediate progenitors (TBR2+) within a 250 μm-width column of E14.5 cortices. WT N=3, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 3, Sas4cKO;Usp28cKO N = 3; one-way ANOVA with post-hoc analysis.(D) Cortices from E14.5 embryos stained with antibodies against Cleaved-Caspase 3 (CC3, white), TP53 (red) and DAPI. Scale bar = 50 μm. (E and F) Quantification of the number of CC3+ apoptotic cells (E) and cells with TP53 activation (F) within a 250 μm-width column of E14.5 cortices. WT N = 5, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 4, Sas4cKO;Usp28cKO N = 4; one-way ANOVA with post-hoc analysis.


Figure 5 ( A ) Representative images of E14 . 5 cortices stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . White and yellow arrows indicate cells with bipolar and monopolar spindles , respectively . Insets showing zoomed in view of 2 representative cells . Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface . Scale bar = 10 μm . ( B ) Percentage of bipolar , monopolar and multipolar cells at the ventricular surface of E14 . 5 cortices . WT n = 201 cells , N = 3 embryos ; Cep63T / T ; Usp28 - / - n = 205 cells , N = 3 embryos ; Sas4cKO ; Usp28cKO n = 308 cells , N = 3 embryos ; # , chi - square test with * , post - hoc analysis , comparisons are made to WT . WT data are from Figure 1F and shown alongside for comparison . ( C ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO ; Usp28cKO N = 3 ; two - tailed Mann - Whitney t - test . 3 WT data points are from Figure 1H and shown alongside for comparison . ( D ) Representative images from live - cell movies of Nuclear - mCherry + WT , Sas4cKO and Sas4cKO ; Usp28cKO NPCs . White arrows showing the nuclei of daughter cells following cytokinesis . ( E ) Plot showing quantification of mitotic duration of WT , Sas4cKO and Sas4cKO ; Usp28cKO NPCs . Triangles represent individual cells ; triangles of the same color represent cells derived from the same embryo . Circles represent the average mitotic duration of cells from each embryo . WT n = 202 cells , N = 4 embryos ; Sas4cKO n = 138 cells , N = 3 embryos ; Sas4cKO ; Usp28cKO n = 191 cells , N = 3 embryos ; two - tailed Welch ' s t - test . WT and Sas4cKO data are from Figure 1K and shown alongside for comparison . ( F ) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO ; Usp28cKO E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 min . Sas4cKO ; Usp28cKO n = 271 cells , N = 3 embryos . ( G ) Percentage of proliferating , arrested and apoptotic progeny within each group of the indicated mitotic duration , calculated from data shown in F ; # , chi - square test with * , post - hoc analysis , comparisons are made to the 0 - 30min group . Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14 . 5 embryos . Each bar represents one cell ; its height represents the amount of time the mother cell spent in mitosis ; bar color represents the fate of the progeny . Dashed line is set at 30 min . ( H and I ) Data from Figures 2C and 2F shown alongside for comparison with F and G .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative images of E14.5 cortices stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). White and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 10 μm.(B) Percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T;Usp28-/- n = 205 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 308 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT. WT data are from Figure 1F and shown alongside for comparison.(C) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T;Usp28-/- N = 3, Sas4cKO;Usp28cKO N = 3; two-tailed Mann-Whitney t-test. 3 WT data points are from Figure 1H and shown alongside for comparison.(D) Representative images from live-cell movies of Nuclear-mCherry+ WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. White arrows showing the nuclei of daughter cells following cytokinesis. (E) Plot showing quantification of mitotic duration of WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Sas4cKO n = 138 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 191 cells, N = 3 embryos; two-tailed Welch's t-test. WT and Sas4cKO data are from Figure 1K and shown alongside for comparison. (F) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO;Usp28cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. Sas4cKO;Usp28cKO n = 271 cells, N = 3 embryos. (G) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in F; #, chi-square test with *, post-hoc analysis, comparisons are made to the 0-30min group.Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. (H and I) Data from Figures 2C and 2F shown alongside for comparison with F and G.


Figure 6 ( a ) WT and Smc5cKO cortices at E14 . 5 stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) and DAPI ( blue ) . Insets showing zoomed in view of 2 representative cells . Scale bar : 25 μm . ( B and C ) Quantification of the number of ( B ) Centrin foci and ( C ) γ - Tubulin foci in mitotic cells in the VZ of E14 . 5 cortices . WT n = 175 cells , N = 4 embryos ; Smc5cKO n = 134 cells , N = 3 embryos . # , chi - square test . WT data are from Figures EV1F and EV1G and shown alongside for comparison . ( D ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) and phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of cells in the VZ and SVZ . Scale bar = 100 μm . ( E ) Ratio of mitotic ( PH3 + ) to cycling ( Ki67 + ) cells in E14 . 5 cortices . WT N = 5 , Smc5cKO N = 5 ; two - tailed Mann - Whitney t - test . WT data are from Figure 1H and shown alongside for comparison . ( F Quantification of the number of ( F ) apoptotic cells ( CC3 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - tailed Mann - Whitney t - test . WT data are from Figures 4E and 4G and shown alongside for comparison . G ) Quantification of the number of ( G ) cells with TP53 activation within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - tailed Mann - Whitney t - test . WT data are from Figures 4E and 4G and shown alongside for comparison . ( H ) Representative whole mount images of WT , Smc5cKO , Smc5cKO ; Usp28cKO , Smc5cKO ; Trp53 - / - brains at P21 . Scale bar = 0 . 2 cm . ( I ) Telencephalon area of P21 brains of the indicated genotypes . WT littermates N = 6 , Smc5cKO N = 5 , Smc5cKO ; Usp28cKO N = 4 , Smc5cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc analysis .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(a) WT and Smc5cKO cortices at E14.5 stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). Insets showing zoomed in view of 2 representative cells. Scale bar: 25 μm. (B and C) Quantification of the number of (B) Centrin foci and (C) γ-Tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Smc5cKO n = 134 cells, N = 3 embryos. #, chi-square test. WT data are from Figures EV1F and EV1G and shown alongside for comparison.(D) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the VZ and SVZ. Scale bar = 100 μm.(E) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Smc5cKO N = 5; two-tailed Mann-Whitney t-test. WT data are from Figure 1H and shown alongside for comparison.(F Quantification of the number of (F) apoptotic cells (CC3+) within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.G) Quantification of the number of (G) cells with TP53 activation within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.(H) Representative whole mount images of WT, Smc5cKO, Smc5cKO;Usp28cKO, Smc5cKO;Trp53-/- brains at P21. Scale bar = 0.2 cm. (I) Telencephalon area of P21 brains of the indicated genotypes. WT littermates N = 6, Smc5cKO N = 5, Smc5cKO;Usp28cKO N = 4, Smc5cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis.