Figure 3 ( A ) Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry - BspF ( grayscale panels ) , incubated on ice with AlexaFluor ™ 488 - Cholera Toxin subunit B ( CTxB ; green ) and shifted to 37 ˚ C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus ( stained using an anti - GM130 antibody ; purple ) . CTxB accumulation within Golgi structures appears white in overlays . Scale bars : 10 and 2 µm ( insets ) . ( B ) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry , mCherry - BspF or HA - BspF over a 30 min time course , expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker , as in ( A ) . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate statistically significant differences compared to mCherry - producing cells as determined by a two - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . ( ( D ) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA - mediated depletion of either Arf6 ( siArf6 ) , Rab8a ( siRab8a ) or Rab6a / a ' ( siRab6a / a ' ) after AlexaFluor ™ 488 - CTxB binding on ice followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . ( E ) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock - infected or infected with wild type ( 2308 ) , ΔbspF , complemented ∆ bspF ( ΔbspF : : bspF ) , ∆ bspB or complemented ∆ bspB ( ΔbspB : : bspB ) bacteria for 24 h , incubated for 30 min with AlexaFluor ™ 488 - CTxB on ice for binding followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate a statistically significant difference compared to mock - infected cells as determined by a one - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . ( ( G - I ) Brucella replication in BMMs treated with non - targeting siRNAs ( siNT ) , or siRNAs against Rab6a / a ' ( siRab6a / a ' ) ( G ) , Rab8a ( siRab8a ) ( H ) or Arf6 ( siArf6 ) ( I ) and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria , measured as number of bacteria per cell at 24 h pi . Data are means ± SD of n = 3 independent experiments in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

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 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 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

Figure 3(A) Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry-BspF (grayscale panels), incubated on ice with AlexaFluor™488-Cholera Toxin subunit B (CTxB; green) and shifted to 37˚C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti-GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). (B) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry-BspF or HA-BspF over a 30 min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared to mCherry-producing cells as determined by a two-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( (D) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA-mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a) or Rab6a/a' (siRab6a/a') after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (E) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock-infected or infected with wild type (2308), ΔbspF, complemented ∆bspF (ΔbspF::bspF), ∆bspB or complemented ∆bspB (ΔbspB::bspB) bacteria for 24 h, incubated for 30 min with AlexaFluor™488-CTxB on ice for binding followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to mock-infected cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( (G-I) Brucella replication in BMMs treated with non-targeting siRNAs (siNT), or siRNAs against Rab6a/a' (siRab6a/a') (G), Rab8a (siRab8a) (H) or Arf6 (siArf6) (I) and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


Figure 4 ( B ) Representative co - immunoprecipitations of HA - BspF and myc - ACAP1 in HeLa cells . HeLa cells were transfected to either co - produce or individually produce HA - BspF and myc - ACAP1 and either HA - BspF or myc - ACAP1 were immunoprecipitated using either anti - HA - conjugated ( upper panel ) or anti - myc - conjugated ( lower panel ) magnetic beads following cross - linking ( + DSP ) or not ( - DSP ) with dithiobis [ succinimidylpropionate ] . Input lysates ( 10 % of the post - nuclear supernatant ) and co - immunoprecipitates were separated by SDS - PAGE and probed for HA - BspF and myc - ACAP1 by Western blotting . ( C ) Representative confocal micrographs of HeLa cells transfected to produce mCherry - BspF and GFP - ACAP1 and treated with Cytochalasin D ( 200 nM ) for 30 min and quantification of colocalization between mCherry - BspF and GFP - ACAP1 . Arrows indicate areas of BspF and ACAP1 colocalization . Scale bars : 10 µm and 2 µm ( insets ) . Data are means ± SD from n = 3 independent experiments in which 10 cells were analyzed per experiment . Pearson ' s correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc _ 2 plug - in .

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 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

Figure 4(B) Representative co-immunoprecipitations of HA-BspF and myc-ACAP1 in HeLa cells. HeLa cells were transfected to either co-produce or individually produce HA-BspF and myc-ACAP1 and either HA-BspF or myc-ACAP1 were immunoprecipitated using either anti-HA-conjugated (upper panel) or anti-myc-conjugated (lower panel) magnetic beads following cross-linking (+DSP) or not (-DSP) with dithiobis[succinimidylpropionate]. Input lysates (10% of the post-nuclear supernatant) and co-immunoprecipitates were separated by SDS-PAGE and probed for HA-BspF and myc-ACAP1 by Western blotting.(C) Representative confocal micrographs of HeLa cells transfected to produce mCherry-BspF and GFP-ACAP1 and treated with Cytochalasin D (200 nM) for 30 min and quantification of colocalization between mCherry-BspF and GFP-ACAP1. Arrows indicate areas of BspF and ACAP1 colocalization. Scale bars: 10 µm and 2 µm (insets). Data are means ± SD from n=3 independent experiments in which 10 cells were analyzed per experiment. Pearson's correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc_2 plug-in.


Figure 5 ( B ) Representative Western blot analysis of co - immunoprecipitations of myc - ACAP1 and Arf6 - HA in the presence or absence of HA - BspF . HeLa cells were transfected to produce Arf6 - HA and combinations of myc - ACAP1 and HA - BspF , or not , and myc - ACAP1 was immunoprecipitated using anti - myc - conjugated magnetic beads . Input lysates ( 6 % of post - nuclear supernatants ) and co - immunoprecipitates were separated by SDS - PAGE and probed for Arf6 - HA , HA - BspF and myc - ACAP1 by Western blotting . Quantification of the Arf6 / ACAP1 ratio was performed by densitometric analysis . Data are means ± SD of 3 independent experiments . The asterisk indicates a statistically significant difference ( P = 0 . 0017 , unpaired Student ' s t test ) between BspF - producing and control conditions . ( E ) Bacterial replication in BMMs transduced to either produce GFP , GFP - ACAP1 or GFP - ACAP1R448Q and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria for 24 h . Data are means ± SD of n = 3 independent experiments , in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , two - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

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 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

Figure 5(B) Representative Western blot analysis of co-immunoprecipitations of myc-ACAP1 and Arf6-HA in the presence or absence of HA-BspF. HeLa cells were transfected to produce Arf6-HA and combinations of myc-ACAP1 and HA-BspF, or not, and myc-ACAP1 was immunoprecipitated using anti-myc-conjugated magnetic beads. Input lysates (6% of post-nuclear supernatants) and co-immunoprecipitates were separated by SDS-PAGE and probed for Arf6-HA, HA-BspF and myc-ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference (P = 0.0017, unpaired Student's t test) between BspF-producing and control conditions.(E) Bacterial replication in BMMs transduced to either produce GFP, GFP-ACAP1 or GFP-ACAP1R448Q and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


Figure 6 ( D - F ) Recruitment of Stx6 - positive vesicles to rBCVs in BMMs treated with non - targeting siRNAs ( siNT ) , siRNAs against Arf6 ( siArf6 ) ( D ) , Rab8a ( siRab8a ) ( E ) or Rab6a / a ' ( siRab6a / a ' ) ( F ) and infected for 24 h with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria . Data are means ± SD of n = 3 independent experiments , in which 200 BCVs were analyzed per experiment . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions ; ns , not significant . ( H ) Brucella replication in BMMs treated with either non - targeting siRNAs ( siNT ) , or siRNAs against Stx6 ( siStx6 ) and infected for 24 h with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria . Data are means ± SD of n = 3 independent experiments , in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

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 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

Figure 6(D-F) Recruitment of Stx6-positive vesicles to rBCVs in BMMs treated with non-targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E) or Rab6a/a' (siRab6a/a') (F) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions; ns, not significant.(H) Brucella replication in BMMs treated with either non-targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


Figure 1A . Immunostaining of P5 cerebellum with indicated antibodies . ( A ) low magnification . ( A ' - A ' ' ' ' ) high magnification . The solid line indicates the pial edge of the EGL . The dashed line demarcates the bottom of the KI67 - stained region . The outer EGL corresponds to the area between the solid and dashed lines , which contain GCPs . Atoh1 KD analysis . ( G , H ) Atoh1 shRNA was introduced to P5 mice EGL together with H2B - EGFP plasmid and immunostaining for NEUROD1 ( Cyan ) and KI67 ( magenta ) was performed 24 hrs later . Nuclei of electroporated cells were labeled with GFP ( green ) . Images of Control ( G ) and Atoh1 KD ( H ) . Arrows indicate the cells of interest where GFP signals are co - localized with NEUROD1 and KI67 . ( I ) Proportions of NEUROD1 + , KI67 + cells ( ND + GCPs ) among the electroporated GCPs ( GFP + , KI67 + cells ) were increased in the Atoh1 KD mice . ( J ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 4 ) and Atoh1 shRNA ( sh # 1 : N = 3 , sh # 2 : N = 3 ) . Bars and error bars are representing as Mean ± SEM . * p < 0 . 05 , N . S . : Not Significant . Number of cells : 98 , 71 , 81 and 60 cells from N = 4 mice ( sclamble ) , 127 , 93 and 51 cells from N = 3 mice ( sh # 1 ) , 75 , 111 , 38 cells from N = 3 mice ( sh # 2 ) .

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 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

Figure 1A. Immunostaining of P5 cerebellum with indicated antibodies. (A) low magnification. (A'-A'''') high magnification. The solid line indicates the pial edge of the EGL. The dashed line demarcates the bottom of the KI67-stained region. The outer EGL corresponds to the area between the solid and dashed lines, which contain GCPs.Atoh1 KD analysis. (G, H) Atoh1 shRNA was introduced to P5 mice EGL together with H2B-EGFP plasmid and immunostaining for NEUROD1 (Cyan) and KI67 (magenta) was performed 24 hrs later. Nuclei of electroporated cells were labeled with GFP (green). Images of Control (G) and Atoh1 KD (H). Arrows indicate the cells of interest where GFP signals are co-localized with NEUROD1 and KI67.(I) Proportions of NEUROD1+, KI67+ cells (ND+GCPs) among the electroporated GCPs (GFP+, KI67+ cells) were increased in the Atoh1 KD mice. (J) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=4) and Atoh1 shRNA (sh#1: N=3, sh#2: N=3). Bars and error bars are representing as Mean ± SEM. *p < 0.05, N.S.: Not Significant. Number of cells: 98,71,81 and 60 cells from N=4 mice (sclamble), 127,93 and 51 cells from N=3 mice (sh#1), 75,111,38 cells from N=3 mice (sh#2).


Figure 2B . Immunostaining of P5 cerebellum with indicated antibodies . Boxplots of the intensity of CCND1 immuno - signal ( B ' ' ' ' , normalized to the mean intensity of AT + GCPs ) . We measured the intensities of CCND1 in ATOH1 + / NEUROD1 - cells and NEUROD1 + / ATOH1 - cells . ATOH1 + / NEUROD1 + cells were not included in this quantification . These boxplots are showing the median ( central band ) , 25 % / 75 % quartiles ( boxes ) and the ranges ( lower whiskers represent the regions of lower box - 1 . 5 * IQR ( inter - quartile range ) , upper whisker represent the regions of upper box - 1 . 5 * IQR ) . Wilcoxon test were performed between AT + GCPs and ND + GCPs . * * * p < 0 . 001 . C - F . Ccnd1 KD analysis . ( C , D ) Ccnd1 shRNAs together with H2B - EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 ( magenta ) and KI67 ( Cyan ) was performed the following day . Nuclei of electroporated cells were labeled with GFP ( yellow ) . Images of Control ( C ) and Ccnd1 KD ( D ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( E ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were decreased in the Ccnd1 KD mice . ( F ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 3 ) and Ccnd1 KD ( sh # 1 : N = 3 , sh # 2 : N = 3 ) mice . Bars and error bars are representing as Mean ± SEM . * * * p < 0 . 001 , N . S . : Not Significant . Number of cells : 52 , 64 and 107 cells from N = 3 mice ( scramble ) , 72 , 79 and 75 cells from N = 3 mice ( sh # 1 ) , 84 , 70 and 40 cells from N = 3 mice ( sh # 2 ) . G - K . Over - expression analysis of Ccnd1 with or without Cdk4 . ( G , H , I ) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 ( magenta ) and KI67 ( cyan ) was performed the following day . Nuclei of electroporated cells were labeled with GFP ( yellow ) . Images of Control ( G ) , CCND1 over expression ( H ) and CCND1 and CDK4 over expression ( I ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( J ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were not significantly changed between Control and CCND1 . Forced expression of CCND1 and CDK4 ( 4D ) significantly increased the proporations of AT + GCPs compared to Control . ( K ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Tukey - Kramer test was performed on Control ( N = 6 ) , CCND1 ( N = 4 ) , CCND1 and CDK4 ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , N . S . : Not Significant . Number of cells : 52 , 64 , 107 , 244 , 127 and 146 from N = 6 mice ( Control ) , 126 , 137 , 80 and 336 from N = 4 mice ( Ccnd1 ) , 212 , 143 and 38 from N = 3 mice ( 4D ) .

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 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 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 O O O O O O O

Figure 2B. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001.C-F. Ccnd1 KD analysis. (C, D) Ccnd1 shRNAs together with H2B-EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (Cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (C) and Ccnd1 KD (D). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (E) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were decreased in the Ccnd1 KD mice. (F) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Bars and error bars are representing as Mean ± SEM. ***p < 0.001, N.S.: Not Significant. Number of cells: 52, 64 and 107 cells from N=3 mice (scramble), 72,79 and 75 cells from N=3 mice (sh#1), 84, 70 and 40 cells from N=3 mice (sh#2).G-K. Over-expression analysis of Ccnd1 with or without Cdk4. (G, H, I) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (G), CCND1 over expression (H) and CCND1 and CDK4 over expression (I). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (J) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were not significantly changed between Control and CCND1. Forced expression of CCND1 and CDK4 (4D) significantly increased the proporations of AT+GCPs compared to Control. (K) Proportions of KI67+ cells (total GCPs) were not significantly changed. Tukey-Kramer test was performed on Control (N=6), CCND1 (N=4), CCND1 and CDK4 (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, N.S.: Not Significant. Number of cells: 52, 64, 107,244,127 and 146 from N=6 mice (Control), 126, 137, 80 and 336 from N=4 mice (Ccnd1), 212, 143 and 38 from N=3 mice (4D).


Figure 3C . N2a cells transfected with Atoh1 - GST plasmids were treated with DMSO ( Control ) , Palbociclib ( Palb ) or Palb and 10 uM MG132 ( MG132 ) for 6hrs before harvest . Lysates of N2a were subjected to immunoblotting with anti - Atoh1 or β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between Control , Palb , MG132 . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * * p < 0 . 001 , * p < 0 . 05 , N . S . : Not Significant . Three slots of each experimental condition represent the three biological replicants of each condition . G . Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids ( 4D ) . N2a cells were treated with 10 nM , 100 nM , 1000 nM Palb for 6 hrs before harvest . Lysates from N2a cells were subjected to immunoblotting with anti - Atoh1 , p - S309 - Ab and β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between 0 , 10 , 100 , 1000nM . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant .

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 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 3C. N2a cells transfected with Atoh1-GST plasmids were treated with DMSO(Control), Palbociclib (Palb) or Palb and 10 uM MG132 (MG132) for 6hrs before harvest. Lysates of N2a were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between Control, Palb, MG132. Bars and error bars are representing as Mean ± SEM from three trials of each condition. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Three slots of each experimental condition represent the three biological replicants of each condition.G. Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids (4D). N2a cells were treated with 10 nM, 100 nM, 1000 nM Palb for 6 hrs before harvest. Lysates from N2a cells were subjected to immunoblotting with anti-Atoh1, p-S309-Ab and β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between 0, 10, 100, 1000nM. Bars and error bars are representing as Mean ± SEM from three trials of each condition. **p < 0.01, *p < 0.05, N.S.: Not Significant.


Figure 4B . Lysates of N2a cells transfected with either WT or a phospho - mimic form of ATOH1 ( S309D ) were immunoblotted with anti - Atoh1 or β - actin . Quantification of relative levels of ATOH1 to the β - ACTIN ( right panel ) . Mean ± s . e . m . Student T - test between WT and S309D was performed . * * * p < 0 . 001 . Three slots of each experimental condition represent the three biological replicants of each condition . Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1 . ( F - I ) Scramble ( Control ) , Atoh1 shRNA ( Atoh1 KD ) , shRNA and WT ( KD + WT ) or shRNA and S309A ( KD + S309A ) plasmids were introduced to P5 mice EGL with H2B - EGFP plasmid and immunostaining for NEUROD1 ( magenta ) was performed at P7 . Nuclei of electroporated cells were detected by GFP ( green ) . Arrows indicate the cells of interest which GFP signals are not co - localized with NEUROD1 ( AT + GCPs ) .

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 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

Figure 4B. Lysates of N2a cells transfected with either WT or a phospho-mimic form of ATOH1 (S309D) were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. ***p < 0.001. Three slots of each experimental condition represent the three biological replicants of each condition.Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (F-I) Scramble (Control), Atoh1 shRNA (Atoh1 KD), shRNA and WT (KD + WT) or shRNA and S309A (KD + S309A) plasmids were introduced to P5 mice EGL with H2B-EGFP plasmid and immunostaining for NEUROD1(magenta) was performed at P7. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest which GFP signals are not co-localized with NEUROD1 (AT+GCPs).


Figure 5B - D . Overexpression analysis of PROX1 . ( B , C ) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected with GFP ( green ) . Images of Control ( B ) , PROX1 overexpression ( C ) . Arrow heads indicates the CCND1 positive cells . White lines are representing as pial surfaces . ( D ) The proportion of CCND1 - positive cells among the electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control ( N = 3 ) and PROX1 overexpression ( N = 3 ) . * * p < 0 . 01 . Number of cells 382 , 147 and 88 cells from N = 3 mice ( control ) , 187 , 108 and 94 cells from N = 3 mice ( Prox1 OE ) . E - G . Prox1 acute KO analysis . ( E , F ) Expression plasmids of Cre recombinase were introduced to P5 EGL of Prox1fl / fl mice and immunostaining for CCND1 ( magenta ) was performed at P8 . Nuclei of electroporated cells were labeled with GFP ( green ) . Images of H2B - GFP electroporation ( E , Control ) , Cre electroporation ( F , Cre ) . Arrows indicate the cells of interest where GFP signals are co - localized with CCND1 . ( G ) The proportions of CCND1 + cells among the electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control ( N = 3 ) and Cre ( N = 3 ) . * * p < 0 . 01 . Number of cells : 238 , 129 and 529 cells from N = 3 mice ( Control ) , 169 , 210 and 216 cells from N = 3 mice ( Cre ) . H - K . Rescue experiment of Prox1 with Ccnd1 and Cdk4 - expressing plasmids . ( H - J ) Prox1 expressing plasmids were introduced with Ccnd1 and Cdk4 expression plasmids ( Prox1 + 4D ) to P5 EGL together with H2B - GFP plasmid . Immunostaining for ATOH1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected by GFP ( green ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( K ) Proportions of ATOH1 + cells ( AT + GCPs ) among the electroporated cells . The Tukey - Kramer test was performed on Control ( N = 6 ) , Prox1 ( N = 3 ) and Prox1 + 4D ( N = 4 ) mice . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Bars and error bars are representing as Mean ± SEM . Control value is the same as Fig 2G , K . Number of cells : 54 , 72 and 59 cells from N = 3 mice ( Prox1 ) , 26 , 53 , 68 and 53 cells from N = 4 mice ( Prox1 + 4D ) . L . ChIP assay in P7 mouse cerebellum with anti - Prox1 or goat IgG as a negative control . Primer set used in this experiment was designed to amplify Ccnd1 promoter region . M - R . Administration of Valproic acid ( VPA ) to electroporated mice . ( M ) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation . Immunostaining for CCND1 ( magenta ) was performed the day following the last VPA treatment . Nuclei of electroporated cells were detected with GFP ( green ) . Arrows indicate the cells of interest where GFP signals are co - localized with CCND1 . ( N - Q ) Images of H2B - GFP with Saline ( N ) , PROX1 with Saline ( O ) , H2B - GFP with VPA ( P ) , and PROX1 with VPA ( Q ) . ( R ) The relative proportions of CCND1 + cells among the electroporated cells ( normalized to the Control ) . Tukey - Kramer test among Control + saline ( N = 4 ) , PROX1 + saline ( N = 3 ) , Control + VPA ( N = 3 ) and PROX1 + VPA ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Number of cells : 120 , 488 , 934 and 211 from N = 4 mice ( Control + saline ) , 630 , 356 and 545 from N = 3 mice ( Control + VPA ) , 363 , 127 and 219 from N = 3 mice ( Prox1 + saline ) , 110 , 116 and 202 from N = 3 mice ( Prox1 + VPA ) .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B-D. Overexpression analysis of PROX1. (B, C) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of Control (B), PROX1 overexpression (C). Arrow heads indicates the CCND1 positive cells. White lines are representing as pial surfaces. (D) The proportion of CCND1-positive cells among the electroporated cells. Bars and error bars are representing as Mean ± SEM. Student T-test between Control (N=3) and PROX1 overexpression (N=3). **p < 0.01. Number of cells 382, 147 and 88 cells from N=3 mice (control), 187, 108 and 94 cells from N=3 mice (Prox1 OE).E-G. Prox1 acute KO analysis. (E, F) Expression plasmids of Cre recombinase were introduced to P5 EGL of Prox1fl/fl mice and immunostaining for CCND1 (magenta) was performed at P8. Nuclei of electroporated cells were labeled with GFP (green). Images of H2B-GFP electroporation (E, Control), Cre electroporation (F, Cre). Arrows indicate the cells of interest where GFP signals are co-localized with CCND1. (G) The proportions of CCND1+ cells among the electroporated cells. Bars and error bars are representing as Mean ± SEM. Student T-test between Control (N=3) and Cre (N=3). **p < 0.01. Number of cells: 238, 129 and 529 cells from N=3 mice (Control), 169, 210 and 216 cells from N=3 mice (Cre).H-K. Rescue experiment of Prox1 with Ccnd1 and Cdk4-expressing plasmids. (H-J) Prox1 expressing plasmids were introduced with Ccnd1 and Cdk4 expression plasmids (Prox1+4D) to P5 EGL together with H2B-GFP plasmid. Immunostaining for ATOH1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (K) Proportions of ATOH1+ cells (AT+GCPs) among the electroporated cells. The Tukey-Kramer test was performed on Control (N=6), Prox1 (N=3) and Prox1+4D (N=4) mice. **p < 0.01, *p <0.05, N.S.: Not Significant. Bars and error bars are representing as Mean ± SEM. Control value is the same as Fig 2G,K. Number of cells: 54, 72 and 59 cells from N=3 mice (Prox1), 26, 53, 68 and 53 cells from N=4 mice (Prox1+4D).L. ChIP assay in P7 mouse cerebellum with anti-Prox1 or goat IgG as a negative control. Primer set used in this experiment was designed to amplify Ccnd1 promoter region. M-R. Administration of Valproic acid (VPA) to electroporated mice. (M) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation. Immunostaining for CCND1 (magenta) was performed the day following the last VPA treatment. Nuclei of electroporated cells were detected with GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with CCND1. (N-Q) Images of H2B-GFP with Saline (N), PROX1 with Saline (O), H2B-GFP with VPA (P), and PROX1 with VPA (Q). (R) The relative proportions of CCND1+ cells among the electroporated cells (normalized to the Control).Tukey-Kramer test among Control+saline (N=4), PROX1+saline (N=3), Control +VPA (N=3) and PROX1 +VPA (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, *p <0.05, N.S.: Not Significant. Number of cells: 120, 488, 934 and 211 from N=4 mice (Control+saline), 630, 356 and 545 from N=3 mice (Control+VPA), 363, 127 and 219 from N=3 mice (Prox1+saline), 110, 116 and 202 from N=3 mice (Prox1+VPA).


Figure 6H - J . Whole cerebellum lysates of P6 , P9 and P12 mice were subjected to immunoblotting with anti - pAtoh1 S309 , Atoh1 , Ccnd1 , Prox1 , β - actin . ( I ) The protein levels of CCND1 and PROX1 normalized by β - ACTIN . ( J ) Quantification of p - L - P . Over - expression analysis of WT or S309D form of Atoh1 . ( L ) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL and immunostaining for KI67 ( magenta ) was performed in P16 mouse cerebellum . Nuclei of electroporated cells were detected by GFP ( green ) . Images of H2B - GFP ( M , Control ) , WT ( N ) and S309D ( O ) . Arrows indicate the cells of interest where GFP signals are co - localized with KI67 . ( GCPs ) . ( P ) Proportions of KI67 + cells among the electroporated cells . Tukey - Kramer test among Control ( N = 3 ) , Atoh1 WT ( N = 3 ) and Atoh1 S309D ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * p < 0 . 05 . Number of cells : 43 , 68 and 46 from N = 3 mice ( Control ) , 104 , 215 and 157 cells from N = 3 mice ( WT ) , 242 , 119 and 111 cells from N = 3 mice ( S309D ) .

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Figure 6H-J. Whole cerebellum lysates of P6, P9 and P12 mice were subjected to immunoblotting with anti-pAtoh1 S309, Atoh1, Ccnd1, Prox1, β-actin. (I) The protein levels of CCND1 and PROX1 normalized by β-ACTIN. (J) Quantification of p-L-P. Over-expression analysis of WT or S309D form of Atoh1. (L) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL and immunostaining for KI67 (magenta) was performed in P16 mouse cerebellum. Nuclei of electroporated cells were detected by GFP (green). Images of H2B-GFP (M, Control), WT(N) and S309D (O). Arrows indicate the cells of interest where GFP signals are co-localized with KI67. (GCPs). (P) Proportions of KI67+ cells among the electroporated cells. Tukey-Kramer test among Control (N=3), Atoh1 WT (N=3) and Atoh1 S309D (N=3). Bars and error bars are representing as Mean ± SEM. *p <0.05. Number of cells: 43, 68 and 46 from N=3 mice (Control), 104, 215 and 157 cells from N=3 mice (WT), 242, 119 and 111 cells from N=3 mice (S309D).


Figure 7D - F . Over - expression analysis of the activated form of β - catenin ( βcat S33Y ) . ( D , E ) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected with GFP ( green ) . Images of H2B - GFP ( D , Control ) and βcat S33Y ( E , S33Y ) . Arrows indicate the cells of interest where GFP signals are co - localized with PROX1 . ( F ) Proportions of PROX1 - positive cells in electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control and S33Y was performed . * * * p < 0 . 001 . Number of cells : 89 , 90 and 55 cells from N = 3 mice ( Control ) , 32 , 111 and 56 cells from N = 3 mice ( S33Y ) . J - L . Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids ( 4D ) to P5 mice EGL and immunostaining for ATOH1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected by GFP ( green ) . Images of βcat S33Y ( J , S33Y ) and βcat S33Y with Ccnd1 and Cdk4 ( K ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . Proportions of ATOH1 + cells ( AT + GCPs ) among the electroporated cells . Control value is the same as Fig 2G - K . Bars and error bars are representing as Mean ± SEM . Tukey - Kramer test was performed between Control , S33Y and S33Y + 4D ( L ) . * * * p < 0 . 001 , N . S : Not Significant . Number of cells : 123 , 138 and 113 cells from N = 3 mice ( S33Y ) , 144 , 223 and 180 cells from N = 3 mice ( S33Y + 4D ) .

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Figure 7D-F. Over-expression analysis of the activated form of β-catenin (βcat S33Y). (D, E) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of H2B-GFP (D, Control) and βcat S33Y (E, S33Y). Arrows indicate the cells of interest where GFP signals are co-localized with PROX1. (F) Proportions of PROX1-positive cells in electroporated cells. Bars and error bars are representing as Mean± SEM. Student T-test between Control and S33Y was performed. ***p < 0.001. Number of cells: 89, 90 and 55 cells from N=3 mice (Control), 32, 111 and 56 cells from N=3 mice (S33Y).J-L. Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids (4D) to P5 mice EGL and immunostaining for ATOH1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Images of βcat S33Y (J, S33Y) and βcat S33Y with Ccnd1 and Cdk4 (K). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1.Proportions of ATOH1+ cells (AT+GCPs) among the electroporated cells. Control value is the same as Fig 2G-K. Bars and error bars are representing as Mean± SEM. Tukey-Kramer test was performed between Control, S33Y and S33Y+4D (L). ***p < 0.001, N.S: Not Significant. Number of cells: 123, 138 and 113 cells from N=3 mice (S33Y), 144, 223 and 180 cells from N=3 mice (S33Y+4D).


Figure 1 ( A ) Coordinates for injection in normal NHPs of the pilot group , study group 1 and study group 2 were calculated on the bases of pre - surgery MRI scans . ( C ) The brain was cut in 6 mm - thick slices using an adjustable brain matrix , obtaining 9 - 10 slices / brain . ( D ) Each brain slice was divided along the midline and each part subdivided in blocks . The cervical spinal cord was cut in 4 blocks .

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Figure 1(A) Coordinates for injection in normal NHPs of the pilot group, study group 1 and study group 2 were calculated on the bases of pre-surgery MRI scans.(C) The brain was cut in 6 mm-thick slices using an adjustable brain matrix, obtaining 9-10 slices/brain. (D) Each brain slice was divided along the midline and each part subdivided in blocks. The cervical spinal cord was cut in 4 blocks.


Figure 2 ( A ) Representative confocal images showing GFP + cells ( green ) expressing markers of neurons ( NeuN ) , astrocytes ( GFAP ) and oligodendrocytes ( CNPase ) close to the posterior injection site of NHP P2 . Arrows indicate co - localization of IF signals . ( B ) Representative confocal images showing neurons ( NeuN ) overexpressing ARSA ( green ) close to the posterior injection site of NHP S2 . 2 . Arrows indicate co - localization of IF signals . Note the granular perinuclear ARSA staining , likely representing protein localized in the Golgi / lysosomal vesicles . ( C ) Confocal z - stack images showing the presence of ARSA - positive cells ( green ) co - stained with oligodendrocyte markers CNPase and APC ( arrows ) . ( D ) Undetectable ARSA immunoreactivity in tissues of untreated animals . In all pictures nuclei are counterstained with ToPro ( blue ) . Scale bars : A , 80 µm ; B , D 60µm , C ,

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Figure 2(A) Representative confocal images showing GFP+ cells (green) expressing markers of neurons (NeuN), astrocytes (GFAP) and oligodendrocytes (CNPase) close to the posterior injection site of NHP P2. Arrows indicate co-localization of IF signals.(B) Representative confocal images showing neurons (NeuN) overexpressing ARSA (green) close to the posterior injection site of NHP S2.2. Arrows indicate co-localization of IF signals. Note the granular perinuclear ARSA staining, likely representing protein localized in the Golgi/lysosomal vesicles.(C) Confocal z-stack images showing the presence of ARSA-positive cells (green) co-stained with oligodendrocyte markers CNPase and APC (arrows). (D) Undetectable ARSA immunoreactivity in tissues of untreated animals. In all pictures nuclei are counterstained with ToPro (blue). Scale bars: A, 80 µm; B, D 60µm, C, 80µm


Figure 3Vector copy number ( VCN ) cartography shows integrated LV genome ( assessed by qPCR ) along the rostro - caudal axis ( slices 1 - 10 ) in LV . hARSA - injected S1 . 1 and S2 . 2 NHP in a side - by - side comparison with ARSA mRNA expression ( assessed by qPCR analyses using probe and primers annealing to sequences conserved in both the human and Macaca fascicularis and expressed as fold increase to region - matched blocks of the contralateral hemisphere ) . Grading of colors for VCN ranged from white ( VCN < 0 . 001 ; corresponding to CT > 37 ) to dark orange ( VCN = 1 - 3 ) . The highest VCN is found in close correspondence to the injection sites , as confirmed by comparison with post - surgery MR images ( yellow and orange circles indicate viral suspension close to the injection sites ) . Grading of colors for ARSA expression ranges from white ( < 1 . 5 fold the physiological level ) to dark purple ( 100 - fold the physiological level ) .

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Figure 3Vector copy number (VCN) cartography shows integrated LV genome (assessed by qPCR) along the rostro-caudal axis (slices 1-10) in LV.hARSA-injected S1.1 and S2.2 NHP in a side-by-side comparison with ARSA mRNA expression (assessed by qPCR analyses using probe and primers annealing to sequences conserved in both the human and Macaca fascicularis and expressed as fold increase to region-matched blocks of the contralateral hemisphere). Grading of colors for VCN ranged from white (VCN<0.001; corresponding to CT>37) to dark orange (VCN=1-3). The highest VCN is found in close correspondence to the injection sites, as confirmed by comparison with post-surgery MR images (yellow and orange circles indicate viral suspension close to the injection sites). Grading of colors for ARSA expression ranges from white (<1.5 fold the physiological level) to dark purple (100-fold the physiological level).


Figure 4 ( A ) ARSA overexpression detected by IHC in cortical and subcortical regions of the injected and contralateral hemispheres of NHP S2 . 3 . ( B ) Physiological ARSA expression in cortical and subcortical regions of the injected hemisphere in LV . GFP - injected NHP P1 . Scale bar , 100 µm . Insets show ARSA - expressing cells at higher magnification . Note the perinuclear accumulation of ARSA protein . ( C ) Quantification of immunopositive signal in brain slices 3 to 6 ( n = 8 blocks / slice ) . Data are expressed as fold increase to mean immunopositive signal measured in matched brain regions of NHP P1 ( physiological ) . Two - way ANOVA and Bonferroni post - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs physiological .

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Figure 4(A) ARSA overexpression detected by IHC in cortical and subcortical regions of the injected and contralateral hemispheres of NHP S2.3.(B) Physiological ARSA expression in cortical and subcortical regions of the injected hemisphere in LV.GFP-injected NHP P1. Scale bar, 100 µm. Insets show ARSA-expressing cells at higher magnification. Note the perinuclear accumulation of ARSA protein.(C) Quantification of immunopositive signal in brain slices 3 to 6 (n= 8 blocks/slice). Data are expressed as fold increase to mean immunopositive signal measured in matched brain regions of NHP P1 (physiological). Two-way ANOVA and Bonferroni post-tests, *p<0.05, **p<0.01, ***p<0.001 vs physiological.


Figure 5 ( A ) Schematic representation of ARSA activity in the brain of NHP S1 . 1 and S2 . 2 along the rostro - caudal axis ( slices 2 - 9 ) . Data are expressed as fold increase to ARSA activity detected in matched blocks of UT and LV . GFP - injected NHPs ( physiological levels ) . Grading of colors ranges from light yellow ( < 1 . 2 fold ) to dark green ( ≥ 2 fold ) . ( B - C ) Enzymatic activity measured in single brain slices of NHPs of study group 1 ( B ) and study group 2 ( C ) ( filled grey bars , injected hemisphere ; striped grey bars , contralateral hemisphere ) , and in UT and LV . GFP - injected NHPs ( white bars ; physiological levels ) . Arrows on x axis indicate the injection sites . n = 2 - 12 tissue blocks / slice . Number of tissue blocks analyzed per animal : 51 ( UT / LV . GFP ) , 98 - 122 ( S1 . 1 and S2 . 2 ) , 12 - 22 ( S1 . 2 , S1 . 3 , S2 . 2 , S2 . 3 ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison tests , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs matched slices of UT / LV . GFP . ( D ) Summary of ARSA activity in the injected and contralateral hemispheres of NHPs in study group 1 and study group 2 . Number of tissue blocks analysed : 51 ( UT / LV . GFP ) , 105 ( study group 1 , injected hemisphere ) , 52 ( study group 1 , contralateral hemisphere ) ; 103 ( study group 2 , injected hemisphere ) , 72 ( study group 2 , contralateral hemisphere ) . One - way ANOVA and Tukey ' s multiple comparison tests , * * * p < 0 . 001 versus UT / LV . GFP , § p < 0 . 05 versus study group 1 . ( E ) ARSA activity in the spinal cord of individual LV . GFP - ( n = 4 blocks / animal , 2 - 3 replicates ) and LV . hARSA - injected NHPs ( n = 2 - 4 blocks / animal ; n = 10 and n = 11 total blocks for study group 1 and study group 2 , respectively ) . Graph on the right represent the summary of ARSA activity in each treatment group . One - way ANOVA and Dunnet ' s multiple comparison test . * p < 0 . 05 versus pilot group . ( F ) ARSA activity in the sciatic nerve of LV . GFP - ( n = 2 ) and LV . hARSA - injected NHPs ( n = 3 ) . In B - F data are expressed as floating bars ( line at mean ) . ( G ) ARSA activity in the cerebrospinal fluid ( CSF ) of UT / LV . GFP ( n = 3 ) and LV . hARSA - injected NHP ( n = 5 ) . Data are expressed as mean ± SEM . Student t - test , * p = 0 . 035 .

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Figure 5(A) Schematic representation of ARSA activity in the brain of NHP S1.1 and S2.2 along the rostro-caudal axis (slices 2-9). Data are expressed as fold increase to ARSA activity detected in matched blocks of UT and LV.GFP-injected NHPs (physiological levels). Grading of colors ranges from light yellow (<1.2 fold) to dark green (≥ 2 fold).(B-C) Enzymatic activity measured in single brain slices of NHPs of study group 1 (B) and study group 2 (C) (filled grey bars, injected hemisphere; striped grey bars, contralateral hemisphere), and in UT and LV.GFP-injected NHPs (white bars; physiological levels). Arrows on x axis indicate the injection sites. n=2-12 tissue blocks/slice. Number of tissue blocks analyzed per animal: 51 (UT/LV.GFP), 98-122 (S1.1 and S2.2), 12-22 (S1.2, S1.3, S2.2, S2.3). Two-way ANOVA followed by Bonferroni's multiple comparison tests, *p<0.05; **p<0.01; ***p<0.001 vs matched slices of UT/LV.GFP.(D) Summary of ARSA activity in the injected and contralateral hemispheres of NHPs in study group 1 and study group 2. Number of tissue blocks analysed: 51 (UT/LV.GFP), 105 (study group 1, injected hemisphere), 52 (study group 1, contralateral hemisphere); 103 (study group 2, injected hemisphere), 72 (study group 2, contralateral hemisphere). One-way ANOVA and Tukey's multiple comparison tests, *** p< 0.001 versus UT/LV.GFP, §p<0.05 versus study group 1.(E) ARSA activity in the spinal cord of individual LV.GFP- (n=4 blocks/animal, 2-3 replicates) and LV.hARSA-injected NHPs (n=2-4 blocks/animal; n=10 and n=11 total blocks for study group 1 and study group 2, respectively). Graph on the right represent the summary of ARSA activity in each treatment group. One-way ANOVA and Dunnet's multiple comparison test. * p<0.05 versus pilot group.(F) ARSA activity in the sciatic nerve of LV.GFP- (n=2) and LV.hARSA-injected NHPs (n=3). In B-F data are expressed as floating bars (line at mean).(G) ARSA activity in the cerebrospinal fluid (CSF) of UT/LV.GFP (n=3) and LV.hARSA-injected NHP (n=5). Data are expressed as mean±SEM. Student t-test, *p=0.035.


Figure 6 ( A ) Vector copy number ( VCN ) indicating the distribution of integrated LV genome along the rostro - caudal axis ( slices 7 - 19 ) in LV . hGALC - injected NHPs ( JT02 and JV02 ) assessed by qPCR . Each dot represent the VCN measured in one block within the slice . Lower threshold ( dotted line ) : VCN < 0 . 001 , corresponding to CT > 37 . ( B ) GALC mRNA expression along the rostro - caudal axis of JT02 ( slices 7 - 13 ) is expressed as fold increase to region - matched blocks of the contralateral hemisphere ( dotted line , y = 1 ) . Data are expressed as floating bars ( min to max , line at mean ; n = 4 - 8 blocks / slice ) . Arrows on x axis in A and B indicate the injection sites . ( C ) Confocal images showing GALC + cells expressing markers of neurons ( NeuN ) and astrocytes ( S100β ) close to the injection site ( slice 10 ) in JT02 . Arrows indicate co - localization of IF signals . ( D - E ) Confocal images showing GALC - expressing astrocytes ( S100β ; D ) and neurons ( NeuN ; E ) in the contralateral hemispheres , anterior ( slice 8 ) and posterior ( slice 12 ) to the injection site . Arrows indicate co - localization of IF signals . Scale bars : C , 70 µm ( upper panel ) and 40µm ( lower panel ) ; D and E , 40µm .

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Figure 6(A) Vector copy number (VCN) indicating the distribution of integrated LV genome along the rostro-caudal axis (slices 7-19) in LV.hGALC-injected NHPs (JT02 and JV02) assessed by qPCR. Each dot represent the VCN measured in one block within the slice. Lower threshold (dotted line): VCN<0.001, corresponding to CT>37.(B) GALC mRNA expression along the rostro-caudal axis of JT02 (slices 7-13) is expressed as fold increase to region-matched blocks of the contralateral hemisphere (dotted line, y=1). Data are expressed as floating bars (min to max, line at mean; n=4-8 blocks/slice). Arrows on x axis in A and B indicate the injection sites.(C) Confocal images showing GALC+ cells expressing markers of neurons (NeuN) and astrocytes (S100β) close to the injection site (slice 10) in JT02. Arrows indicate co-localization of IF signals.(D-E) Confocal images showing GALC-expressing astrocytes (S100β; D) and neurons (NeuN; E) in the contralateral hemispheres, anterior (slice 8) and posterior (slice 12) to the injection site. Arrows indicate co-localization of IF signals. Scale bars: C, 70 µm (upper panel) and 40µm (lower panel); D and E, 40µm.


Figure 7 ( A ) Specific GALC enzymatic activity in single brain slices of WT untreated ( UT ; white bars , physiological levels ) , WT LV . hGALC - treated ( JV02 ) and Krabbe - affected LV . hGALC - treated ( JV02 ) animals ( filled grey bars , injected hemisphere ; striped grey bars , contralateral hemisphere ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison tests ; * p < 0 . 05 , * * p < 0 . 01 vs matched slices of WT UT . ( B - C ) Summary of GALC activity in brain ( B ) samples from JT02 and JV02 , as well as from WT and Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , line at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / animal : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis test and Dunn ' s multiple comparison test ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT . ( B - C ) Summary of GALC activity in spinal cord ( C ; n = 3 blocks / animal ) samples from JT02 and JV02 , as well as from WT and Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , line at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / animal : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis test and Dunn ' s multiple comparison test ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT . ( D ) Chromatographic profile of GALC in selected brain slices and spinal cord ( SC ) tissue of untreated ( UT ) WT and LV . hGALC - treated Krabbe NHP ( JT02 ) . Extracts were run through a Sephadex S - 300 gel filtration column . Fractions ( 0 . 2 ml ) were assayed for enzyme activity using MUGAL substrate in the presence ( ) or absence of 11µM AgNO3 ( ● ) . ( E ) GALC activity in the CSF of JT02 ( pre - and post - treatment ) and JV02 ( post - treatment ) . Dots represent technical replicates . ( F ) Beginning at two months ( m ) of age , animals were evaluated monthly for motor performance using a modified Bayley ' s scale for infant development . For the study animals ( JV02 and JT02 ) , scores are reported as pre - surgery and post - surgery . Historical data for normal and affected animals are presented as means plus one standard deviation . Affected animals score significantly lower than normal animals at any age considered . JT02 ' s motor scores increase over time post LV . GALC injection , being close to the mean observed in normal juveniles at 5 months . Historical data are analyzed by two - way ANOVA and Bonferroni posttests , p < 0 . 05 at 6m , p < 0 . 001 at all other ages . n = 5 - 20 animals / group , except for normal animals at 6 months ( n = 1 ) .

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Figure 7(A) Specific GALC enzymatic activity in single brain slices of WT untreated (UT; white bars, physiological levels), WT LV.hGALC-treated (JV02) and Krabbe-affected LV.hGALC-treated (JV02) animals (filled grey bars, injected hemisphere; striped grey bars, contralateral hemisphere). Two-way ANOVA followed by Bonferroni's multiple comparison tests; *p<0.05, **p<0.01 vs matched slices of WT UT.(B-C) Summary of GALC activity in brain (B) samples from JT02 and JV02, as well as from WT and Krabbe UT controls. Data in A-C are represented as floating bars (min to max, line at mean; n=2-6 blocks/slice). Number of blocks analysed/animal: 16 (WT UT), 24 (JT02), 22 (JV02). GALC activity in Krabbe UT samples is < 0.001 nmol/h*mg; n=3 blocks. Kruskal-Wallis test and Dunn's multiple comparison test; *p<0.05, ***p<0.001 vs WT UT.(B-C) Summary of GALC activity in spinal cord (C; n=3 blocks/animal) samples from JT02 and JV02, as well as from WT and Krabbe UT controls. Data in A-C are represented as floating bars (min to max, line at mean; n=2-6 blocks/slice). Number of blocks analysed/animal: 16 (WT UT), 24 (JT02), 22 (JV02). GALC activity in Krabbe UT samples is < 0.001 nmol/h*mg; n=3 blocks. Kruskal-Wallis test and Dunn's multiple comparison test; *p<0.05, ***p<0.001 vs WT UT.(D) Chromatographic profile of GALC in selected brain slices and spinal cord (SC) tissue of untreated (UT) WT and LV.hGALC-treated Krabbe NHP (JT02). Extracts were run through a Sephadex S-300 gel filtration column. Fractions (0.2 ml) were assayed for enzyme activity using MUGAL substrate in the presence () or absence of 11µM AgNO3 (●).(E) GALC activity in the CSF of JT02 (pre- and post-treatment) and JV02 (post-treatment). Dots represent technical replicates.(F) Beginning at two months (m) of age, animals were evaluated monthly for motor performance using a modified Bayley's scale for infant development. For the study animals (JV02 and JT02), scores are reported as pre-surgery and post-surgery. Historical data for normal and affected animals are presented as means plus one standard deviation. Affected animals score significantly lower than normal animals at any age considered. JT02's motor scores increase over time post LV.GALC injection, being close to the mean observed in normal juveniles at 5 months. Historical data are analyzed by two-way ANOVA and Bonferroni posttests, p<0.05 at 6m, p<0.001 at all other ages. n=5-20 animals/group, except for normal animals at 6 months (n=1).


Figure 1E . Ribosomal pull down shows lack of occupancy of ribosomes on veal2 . fli1a and actb were used as positive controls . F - I . e - GFP fusion assay confirms lack of peptide formation from veal2 sequence . ( F - G ) mitfa - eGFP fused transcript . ( H - I ) veal2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected embryos . Scale bar - 100μm .

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Figure 1E. Ribosomal pull down shows lack of occupancy of ribosomes on veal2. fli1a and actb were used as positive controls.F-I. e-GFP fusion assay confirms lack of peptide formation from veal2 sequence. (F-G) mitfa-eGFP fused transcript. (H-I) veal2-eGFP fused transcript. Arrowheads indicating e-GFP expression in mitfa-eGFP injected embryos. Scale bar-100μm.


Figure 2F - T . Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2gib005Δ8 / + zebrafish upon complementing with the wild type ( WT ) veal2 RNA . ( F - J ) Control zebrafish embryos . ( K - O ) veal2gib005Δ8 / + embryos injected with vehicle control . ( P - T ) veal2gib005Δ8 / + embryos complemented with veal2 RNA . ( F , K , P ) bright field . ( G , L , Q ) mRFP . ( H , M , R ) Animals stained with O - dianisidine stain . ( I , N , S ) eGFP . ( J , O , T ) merged eGFP and mRFP filters . Arrowheads show the presence of hemorrhage due to the vascular integrity defects . ( F - H , K - M , P - R ) magnification - 5X , scale bar - 100μm ( I - J , N - O , S - T ) magnification - 20X , Scale bar - 50μm .

Figure 2F-T. Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2gib005Δ8/+ zebrafish upon complementing with the wild type (WT) veal2 RNA. (F-J) Control zebrafish embryos. (K-O) veal2gib005Δ8/+ embryos injected with vehicle control. (P-T) veal2gib005Δ8/+ embryos complemented with veal2 RNA. (F,K,P) bright field. (G,L,Q) mRFP. (H,M,R) Animals stained with O-dianisidine stain. (I,N,S) eGFP. (J,O,T) merged eGFP and mRFP filters. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (F-H,K-M,P-R) magnification-5X, scale bar-100μm (I-J,N-O,S-T) magnification-20X, Scale bar-50μm.


Figure 3C . RNA immunoprecipitation of veal2 was performed by pull - down of Prkcbb and Epbhb3 , followed by qRT - PCR . IgG pull - down was performed as control . Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples , along with 10 % input control , as marked by arrowheads . D . qRT - PCR based quantification of veal2 transcript across Prkcbb , Ephb3 and IgG immunoprecipitations . actb was used as normalization control . Data from three different biological replicates represented as mean fold change values ± standard deviation . F - K . Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8 / + zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8 / + . Arrowheads show the presence of hemorrhage due to the vascular integrity defects . Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure . ( F - I ) Magnification - 5X , Scale bar - 100μm , ( J - K ) Magnification - 20X , Scale bars - 50μm .

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Figure 3C. RNA immunoprecipitation of veal2 was performed by pull-down of Prkcbb and Epbhb3, followed by qRT-PCR. IgG pull-down was performed as control. Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples, along with 10% input control, as marked by arrowheads.D. qRT-PCR based quantification of veal2 transcript across Prkcbb, Ephb3 and IgG immunoprecipitations. actb was used as normalization control. Data from three different biological replicates represented as mean fold change values ± standard deviation.F-K. Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8/+ zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8/+. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure. (F-I) Magnification-5X, Scale bar-100μm, (J-K) Magnification-20X, Scale bars-50μm.


Figure 4F . Abundance of Prkcbb protein in total cell ( T ) , cytoplasmic ( C ) and membrane ( M ) fractions of cells from 2 dpf gib004Tg ( fli1a : EGFP ; gata1a : DsRed ) and veal2gib005Δ8 / + zebrafish embryos . Arrowhead indicates Prkcbb enrichment in the membrane fraction of veal2gib005Δ8 / + zebrafish embryos .

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Figure 4F. Abundance of Prkcbb protein in total cell (T), cytoplasmic (C) and membrane (M) fractions of cells from 2 dpf gib004Tg(fli1a:EGFP;gata1a:DsRed) and veal2gib005Δ8/+ zebrafish embryos. Arrowhead indicates Prkcbb enrichment in the membrane fraction of veal2gib005Δ8/+ zebrafish embryos.


Figure 5E - H e - GFP fusion assay confirms lack of peptide formation from veal2 sequence . ( E - F ) mitfa - eGFP fused transcript . ( G - H ) VEAL2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected embryos . Scale bar - 100μI - N . Complementation of VEAL2 in veal2gib005Δ8 / + embryos significantly rescued hemorrhage phenotype . Arrowheads show the presence of hemorrhage due to the vascular integrity defects . ( I - L ) Magnification - 5X , Scale bar - 100μm ( M - N ) Magnification - 20X , Scale bars - 50μm . R - T . Co IF for PRKCB and smFISH of VEAL2 highlight their colocalization . ( R ) PRKCB in the GFP channel . ( S ) VEAL2 in CAL - fluor red ( 610nM ) . ( T ) Merged image for PRKCB and VEAL2 . Magnification - 100X , scale bar - 20μm .

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Figure 5E-H e-GFP fusion assay confirms lack of peptide formation from veal2 sequence. (E-F) mitfa-eGFP fused transcript. (G-H) VEAL2-eGFP fused transcript. Arrowheads indicating e-GFP expression in mitfa-eGFP injected embryos. Scale bar-100μI-N. Complementation of VEAL2 in veal2gib005Δ8/+ embryos significantly rescued hemorrhage phenotype. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (I-L) Magnification-5X, Scale bar-100μm (M-N) Magnification-20X, Scale bars-50μm.R-T. Co IF for PRKCB and smFISH of VEAL2 highlight their colocalization. (R) PRKCB in the GFP channel. (S) VEAL2 in CAL-fluor red (610nM). (T) Merged image for PRKCB and VEAL2. Magnification-100X, scale bar-20μm.


Figure 6K - AB . VEAL2 regulates junctional dynamics by interacting with PRKCB . Overexpression of VEAL2 retains PRKCB mostly in cytoplasm and keeps strong junctional assembly formation of CDH5 and CTNNB1 on the membrane . Knockdown of VEAL2 led to migration of PRKCB on membrane and henceforth degradation of junctional assembly of CDH5 and CTNNB1 . ( K - N , W - X ) PRKCB . ( O - R , Y - Z ) CDH5 . ( S - V , AA - AB ) CTNNB1 . ( K - V ) Magnification - 60X , Scale bar - 15μm . Arrowheads indicate representation of signals of proteins in HUVECs . ( W - AB ) Dot plot representing quantification of protein signal localization in membrane / total fraction . The quantification was done using imageJ . Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation . Data is shown as individual values ; the middle bar represents the mean and the error bar represents ± standard deviation .

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Figure 6K-AB. VEAL2 regulates junctional dynamics by interacting with PRKCB. Overexpression of VEAL2 retains PRKCB mostly in cytoplasm and keeps strong junctional assembly formation of CDH5 and CTNNB1 on the membrane. Knockdown of VEAL2 led to migration of PRKCB on membrane and henceforth degradation of junctional assembly of CDH5 and CTNNB1. (K-N,W-X) PRKCB. (O-R,Y-Z) CDH5. (S-V,AA-AB) CTNNB1. (K-V) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (W-AB) Dot plot representing quantification of protein signal localization in membrane/total fraction. The quantification was done using imageJ. Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.


Figure 7E - S . Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein . Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia . ( E - H , Q ) CDH5 protein , ( I - L , R ) CTNNB1 protein , ( M - P , S ) PRKCB protein . ( E - P ) Magnification - 60X , Scale bar - 15μm . Arrowheads indicate representation of signals of proteins in HUVECs . ( Q - S ) Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation . Data is shown as individual values ; the middle bar represents the mean and the error bar represents ± standard deviation .

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Figure 7E-S. Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E-H,Q) CDH5 protein, (I-L,R) CTNNB1 protein, (M-P,S) PRKCB protein. (E-P) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q-S) Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.


Figure 1 C Wild - type and tel1 ∆ strains expressing a fully functional Top1 - HA tagged protein were arrested in G1 with α - factor ( αf ) and released into YEPD supplemented with CPT ( 50 μM ) . Western blot with anti - HA antibodies ( top ) and Coomassie staining ( bottom ) of protein extracts prepared at the indicated time points

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Figure 1 C Wild-type and tel1∆ strains expressing a fully functional Top1-HA tagged protein were arrested in G1 with α-factor (αf) and released into YEPD supplemented with CPT (50 μM). Western blot with anti-HA antibodies (top) and Coomassie staining (bottom) of protein extracts prepared at the indicated time points


Figure 3 A - C Exponentially growing cell cultures ( exp ) were arrested in G1 with α - factor ( αf ) and released into YEPD with or without CPT ( 50 μM ) . Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti - Rad53 antibodies ( C ) E , F G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) ( E ) or in YEPD supplemented with CPT ( 10 μM ) with ( + noc ) or without ( - noc ) nocodazole ( F ) . Western blot analysis with anti - Rad53 antibodies

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Figure 3 A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti-Rad53 antibodies (C) E, F G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM) (E) or in YEPD supplemented with CPT (10 μM) with (+noc) or without (-noc) nocodazole (F). Western blot analysis with anti-Rad53 antibodies


Figure 4 A - C Exponentially growing cell cultures were arrested in G1 with α - factor ( αf ) and released in YEPD supplemented with CPT ( 50 μM ) . ( A ) Western blot analysis with anti - Rad53 antibodies

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Figure 4 A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM). (A) Western blot analysis with anti-Rad53 antibodies


Figure 5 ( B - E ) G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at the indicated time points were subjected to western blot analysis with anti - Rad53 antibodie ( B - E ) G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at the indicated time points were subjected to western blot analysis with anti - Rad53 antibodies ( B , D

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Figure 5 (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodie  (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodies (B, D


Figure 6 F , G G1 - arrested cell cultures ( αf ) were released into YEPD supplemented with CPT ( 50 μM ) . Western blot analysis with anti - Rad53 antibodies

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Figure 6 F, G G1-arrested cell cultures (αf) were released into YEPD supplemented with CPT (50 μM). Western blot analysis with anti-Rad53 antibodies


Figure 1HeLa and cyclin F Knock - Out ( CCNF K / O ) cells were treated with Kinase ChemoGenomic Set ( KSGS ) in 384 well format . After 72 Hours ( h ) viability was measured using rezasurin . Flowchart representation ( left ) . Robust Z - score difference is plotted for data acquired at 1 μM concentration ( right ) . Cell survival measured using rezasurin and compared to controls treated with DMSO ( expressed as relative proliferation % ) . Cells were treated with the indicated inhibitors at 0 . 1 and 1 μM concentration . Cell survival measured using rezasurin and compared to controls ( 0 ) treated with DMSO ( expressed as relative proliferation % ) . Cells were treated with UCN - 01 at the indicated concentrations . Differences in viability of HeLa and CCNF K / O cells after treatment with specific Chk1i ( LY2603618 ) at the indicated concentrations plotted on a log10 scale to measure differences in IC50 . Number of cells was measured in HeLa and CCNF K / O cells left untreated or treated with Chk1i ( LY2603618 ) for 24h , 48h , or 72h as indicated . ( n . s . = non - significant ) . Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K / O cells after treatment with Chk1i ( LY2603618 ) . U - 2 - OS cells were seeded and transfected with non - targeting siRNA siNC ( Negative Control ) or siCyc F . 24 hours after transfection , cells were treated with UCN - 01 , or Chk1i ( LY2603618 ) at indicated concentrations for 3 days before viability was measured . Cell viability measurements using Propidium Iodide staining in U - 2 - OS cells transfected with non - targeting siNC and siCyc F and treated with Chk1i ( LY2603618 ) as indicated .

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 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 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 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 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 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

Figure 1HeLa and cyclin F Knock-Out (CCNF K/O) cells were treated with Kinase ChemoGenomic Set (KSGS) in 384 well format. After 72 Hours (h) viability was measured using rezasurin. Flowchart representation (left). Robust Z-score difference is plotted for data acquired at 1 μM concentration (right).Cell survival measured using rezasurin and compared to controls treated with DMSO (expressed as relative proliferation %). Cells were treated with the indicated inhibitors at 0.1 and 1 μM concentration.Cell survival measured using rezasurin and compared to controls (0) treated with DMSO (expressed as relative proliferation %). Cells were treated with UCN-01 at the indicated concentrations.Differences in viability of HeLa and CCNF K/O cells after treatment with specific Chk1i (LY2603618) at the indicated concentrations plotted on a log10 scale to measure differences in IC50.Number of cells was measured in HeLa and CCNF K/O cells left untreated or treated with Chk1i (LY2603618) for 24h, 48h, or 72h as indicated. (n.s. = non-significant).Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K/O cells after treatment with Chk1i (LY2603618).U-2-OS cells were seeded and transfected with non-targeting siRNA siNC (Negative Control) or siCyc F. 24 hours after transfection, cells were treated with UCN-01, or Chk1i (LY2603618) at indicated concentrations for 3 days before viability was measured.Cell viability measurements using Propidium Iodide staining in U-2-OS cells transfected with non-targeting siNC and siCyc F and treated with Chk1i (LY2603618) as indicated.


Figure 2Cells treated with 1μM Chk1i ( LY2603618 ) for the indicated hours ( h ) were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by Western blot ( WB ) . TFIIH was used as a loading control . Percentage ( % ) of γ - H2AX positive cells in G1 cells after Chk1i ( LY2603618 ) treatment for the indicated hours ( h ) measured using Fluorescence Activated Cell Sorting ( FACS ) . G1 cells were considered having DAPI 2n staining and EdU negative . Percentage ( % ) of γ - H2AX positive cells in replicating ( EdU + ) cells after Chk1i ( LY2603618 ) treatment at the indicated time points . Percentage ( % ) of γ - H2AX positive cells in G2 / M cells after Chk1i ( LY2603618 ) treatment . G2 / M cells were considered having DAPI 4n staining and EdU negativity . Intensity of γ - H2AX measured as Relative Fluorescence Units ( RFU ) in early S phase cells treated with Chk1i . Mean intensity of γ - H2AX fluorescence was evaluated from EdU + cells with 2N DAPI content . Intensity of γ - H2AX measured as Relative Fluorescence Units ( RFU ) in replicating cells treated with Chk1i . Mean intensity of γ - H2AX fluorescence was evaluated from EdU + . Overall , cell cycle profile of HeLa and CCNF K / O cells after Chk1i treatment . Cells with 2n DAPI staining but EdU - were gated as G1 cells ; cells with > 2n but < 4n DAPI staining but EdU - were gated as EdU - S cells ; cells with > 2n but < 4n DAPI staining but EdU + were gated as EdU + S phase ; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells . Data from at least 3 independent replicates and plotted as Mean ± SD . Representative plots of γ - H2AX vs RPA2 signal of HeLa and CCNF K / O cells after treatment with 1μM Chk1i ( LY2603618 ) at the indicated hours ( h ) . Blue labeled dots represent γ - H2AX positive cells . Relates to materials section for details on representation .

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 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 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 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

Figure 2Cells treated with 1μM Chk1i (LY2603618) for the indicated hours (h) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by Western blot (WB). TFIIH was used as a loading control.Percentage (%) of γ-H2AX positive cells in G1 cells after Chk1i (LY2603618) treatment for the indicated hours (h) measured using Fluorescence Activated Cell Sorting (FACS). G1 cells were considered having DAPI 2n staining and EdU negative. Percentage (%) of γ-H2AX positive cells in replicating (EdU+) cells after Chk1i (LY2603618) treatment at the indicated time points. Percentage (%) of γ-H2AX positive cells in G2/M cells after Chk1i (LY2603618) treatment. G2/M cells were considered having DAPI 4n staining and EdU negativity.Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in early S phase cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+ cells with 2N DAPI content. Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in replicating cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+. Overall, cell cycle profile of HeLa and CCNF K/O cells after Chk1i treatment. Cells with 2n DAPI staining but EdU- were gated as G1 cells; cells with >2n but <4n DAPI staining but EdU- were gated as EdU- S cells; cells with >2n but <4n DAPI staining but EdU+ were gated as EdU+ S phase; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells. Data from at least 3 independent replicates and plotted as Mean ± SD.Representative plots of γ-H2AX vs RPA2 signal of HeLa and CCNF K/O cells after treatment with 1μM Chk1i (LY2603618) at the indicated hours (h). Blue labeled dots represent γ-H2AX positive cells. Relates to materials section for details on representation.


Figure 3Representative plots of γ - H2AX intensity vs DAPI signal of CCNF K / O cells transfected with empty GFP and GFP - CCNF untreated ( top panels ) and after treatment with 1μM Chk1i ( LY2603618 ) for 20h ( bottom panels ) . Normalised γ - H2AX intensity in CCNF K / O cells transfected with either GFP empty vector or GFP - Cyc F and treated with 1μM Chk1i for 20 hours . GFP positive cells were gated . Data from 3 independent experiments were plotted with Mean % ± SD . Representative images of neutral and alkaline comet assay from HeLa and CCNF K / O cells treated with Chk1i . Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated . Quantification of comets in alkaline gels . At least 100 cells , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) and 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using the Mann - Whitney test ( two - tailed ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % DNA / 100 . Quantification of comets in neutral gels . At least 100 cells , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) and 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using the Mann - Whitney test ( two - tailed ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % DNA / 100 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Representative plots of γ-H2AX intensity vs DAPI signal of CCNF K/O cells transfected with empty GFP and GFP-CCNF untreated (top panels) and after treatment with 1μM Chk1i (LY2603618) for 20h (bottom panels). Normalised γ-H2AX intensity in CCNF K/O cells transfected with either GFP empty vector or GFP-Cyc F and treated with 1μM Chk1i for 20 hours. GFP positive cells were gated. Data from 3 independent experiments were plotted with Mean % ± SD.Representative images of neutral and alkaline comet assay from HeLa and CCNF K/O cells treated with Chk1i. Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated. Quantification of comets in alkaline gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100. Quantification of comets in neutral gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100.