G - K . Over - expression of Ccnd1 with or without Cdk4 . ( G , H , I ) Ccnd1 Cdk4 expression plasmids were introduced to P5 mice EGL immunostaining for ATOH1 ( magenta ) KI67 ( cyan ) was performed following day . Nuclei of electroporated were labeled with GFP ( yellow ) . of Control ( G ) , CCND1 over expression ( H ) CCND1 CDK4 over expression ( I ) . Arrows indicate of interest where GFP signals are co - localized with ATOH1 . ( J ) Proportions of ATOH1 + , KI67 + ( AT + GCPs ) among electroporated were not significantly changed between Control CCND1 . Forced expression of CCND1 CDK4 ( 4D ) significantly increased proporations of AT + GCPs compared to Control . ( K ) Proportions of KI67 + ( total GCPs ) were not significantly changed . Tukey - Kramer was performed on Control ( N = 6 ) , CCND1 ( N = 4 ) , CCND1 CDK4 ( N = 3 ) . Bars error bars are representing as Mean ± . * * p < 0 . 01 , N . S . : Not Significant . Number of : 52 , 64 , 107 , 244 , 127 146 from N = 6 mice ( Control ) , 126 , 137 , 80 336 from N = 4 mice ( Ccnd1 ) , 212 , 143 38 from N = 3 mice ( 4D ) .

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

G-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).


C . N2a transfected with Atoh1 - GST plasmids were treated with DMSO ( Control ) , Palbociclib ( Palb ) or Palb 10 uM MG132 ( MG132 ) for 6hrs before harvest . Lysates of N2a were subjected to immunoblotting with anti - Atoh1 or β - actin . of relative ATOH1 to β - ACTIN ( right panel ) . Tukey - Kramer was performed between Control , Palb , MG132 . Bars error bars are representing as Mean ± from three trials of each condition . * * * p < 0 . 001 , * p < 0 . 05 , N . S . : Not Significant . Three slots of each experimental condition represent three biological replicants of each condition .

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

C. 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 with Ccnd1 Cdk4 expression plasmids ( 4D ) . N2a were treated with 10 nM , 100 nM , 1000 nM Palb for 6 hrs before harvest . Lysates from N2a were subjected to immunoblotting with anti - Atoh1 , p - S309 - Ab β - actin . of relative ATOH1 to β - ACTIN ( right panel ) . Tukey - Kramer was performed between 0 , 10 , 100 , 1000nM . Bars error bars are representing as Mean ± from three trials of each condition . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant .

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

G. 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.


B . Lysates of N2a transfected with either WT or a phospho - mimic form of ATOH1 ( S309D ) were immunoblotted with anti - Atoh1 or β - actin . of relative of ATOH1 to β - ACTIN ( right panel ) . Mean ± s . e . m . Student T - between WT S309D was performed . * * * p < 0 . 001 . Three slots of each experimental condition represent three biological replicants of each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 WT ( KD + WT ) or shRNA S309A ( KD + S309A ) plasmids were introduced to P5 mice EGL with H2B - EGFP plasmid immunostaining for NEUROD1 ( magenta ) was performed at P7 . Nuclei of electroporated were detected by GFP ( green ) . Arrows indicate of interest which GFP signals are not co - localized with NEUROD1 ( AT + GCPs ) .

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

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).


B - D . Overexpression of PROX1 . ( B , C ) Prox1 expression plasmids were introduced to mouse P5 EGL immunostaining for CCND1 ( magenta ) was performed following day . Nuclei of electroporated were detected with GFP ( green ) . of Control ( B ) , PROX1 overexpression ( C ) . Arrow heads indicates CCND1 positive . lines are representing as pial surfaces . ( D ) proportion of CCND1 - positive among electroporated . Bars error bars are representing as Mean ± . Student T - between Control ( N = 3 ) PROX1 overexpression ( N = 3 ) . * * p < 0 . 01 . Number of 382 , 147 88 from N = 3 mice ( control ) , 187 , 108 94 from N = 3 mice ( Prox1 OE ) .

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

B-D. 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 . ( E , F ) Expression plasmids of Cre recombinase were introduced to P5 EGL of Prox1fl / fl mice immunostaining for CCND1 ( magenta ) was performed at P8 . Nuclei of electroporated were labeled with GFP ( green ) . of H2B - GFP electroporation ( E , Control ) , Cre electroporation ( F , Cre ) . Arrows indicate of interest where GFP signals are co - localized with CCND1 . ( G ) proportions of CCND1 + among electroporated . Bars error bars are representing as Mean ± . Student T - between Control ( N = 3 ) Cre ( N = 3 ) . * * p < 0 . 01 . Number of : 238 , 129 529 from N = 3 mice ( Control ) , 169 , 210 216 from N = 3 mice ( Cre ) .

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

E-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 Cdk4 - expressing plasmids . ( H - J ) Prox1 expressing plasmids were introduced with Ccnd1 Cdk4 expression plasmids ( Prox1 + 4D ) to P5 EGL together with H2B - GFP plasmid . Immunostaining for ATOH1 ( magenta ) was performed following day . Nuclei of electroporated were detected by GFP ( green ) . Arrows indicate of interest where GFP signals are co - localized with ATOH1 . ( K ) Proportions of ATOH1 + ( AT + GCPs ) among electroporated . Tukey - Kramer was performed on Control ( N = 6 ) , Prox1 ( N = 3 ) Prox1 + 4D ( N = 4 ) mice . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Bars error bars are representing as Mean ± . Control value is same as Fig 2G , K . Number of : 54 , 72 59 from N = 3 mice ( Prox1 ) , 26 , 53 , 68 53 from N = 4 mice ( Prox1 + 4D ) .

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

H-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 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 VPA or Saline was administered intraperitoneally 2 days after electroporation . Immunostaining for CCND1 ( magenta ) was performed day following last VPA treatment . Nuclei of electroporated were detected with GFP ( green ) . Arrows indicate of interest where GFP signals are co - localized with CCND1 . ( N - Q ) of H2B - GFP with Saline ( N ) , PROX1 with Saline ( O ) , H2B - GFP with VPA ( P ) , PROX1 with VPA ( Q ) . ( R ) relative proportions of CCND1 + among electroporated ( normalized to Control ) . Tukey - Kramer among Control + saline ( N = 4 ) , PROX1 + saline ( N = 3 ) , Control + VPA ( N = 3 ) PROX1 + VPA ( N = 3 ) . Bars error bars are representing as Mean ± . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Number of : 120 , 488 , 934 211 from N = 4 mice ( Control + saline ) , 630 , 356 545 from N = 3 mice ( Control + VPA ) , 363 , 127 219 from N = 3 mice ( Prox1 + saline ) , 110 , 116 202 from N = 3 mice ( Prox1 + VPA ) .

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

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).


H - J . Whole cerebellum lysates of P6 , P9 P12 mice were subjected to immunoblotting with anti - pAtoh1 S309 , Atoh1 , Ccnd1 , Prox1 , β - actin . ( I ) of CCND1 PROX1 normalized by β - ACTIN . ( J ) of p -

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

H-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 of WT or S309D form of Atoh1 . ( L ) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL immunostaining for KI67 ( magenta ) was performed in P16 mouse cerebellum . Nuclei of electroporated were detected by GFP ( green ) . of H2B - GFP ( M , Control ) , WT ( N ) S309D ( O ) . Arrows indicate of interest where GFP signals are co - localized with KI67 . ( GCPs ) . ( P ) Proportions of KI67 + among electroporated . Tukey - Kramer among Control ( N = 3 ) , Atoh1 WT ( N = 3 ) Atoh1 S309D ( N = 3 ) . Bars error bars are representing as Mean ± . * p < 0 . 05 . Number of : 43 , 68 46 from N = 3 mice ( Control ) , 104 , 215 157 from N = 3 mice ( WT ) , 242 , 119 111 from N = 3 mice ( S309D ) .

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

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).


D - F . Over - expression of activated form of β - catenin ( βcat S33Y ) . ( D , E ) Expression plasmids of βcat S33Y were introduced to P5 mice EGL immunostaining for PROX1 ( magenta ) was performed following day . Nuclei of electroporated were detected with GFP ( green ) . of H2B - GFP ( D , Control ) βcat S33Y ( E , S33Y ) . Arrows indicate of interest where GFP signals are co - localized with PROX1 . ( F ) Proportions of PROX1 - positive in electroporated . Bars error bars are representing as Mean ± . Student T - between Control S33Y was performed . * * * p < 0 . 001 . Number of : 89 , 90 55 from N = 3 mice ( Control ) , 32 , 111 56 from N = 3 mice ( S33Y ) .

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

D-F. 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 Cdk4 expression plasmids ( 4D ) to P5 mice EGL immunostaining for ATOH1 ( magenta ) was performed following day . Nuclei of electroporated were detected by GFP ( green ) . of βcat S33Y ( J , S33Y ) βcat S33Y with Ccnd1 Cdk4 ( K ) . Arrows indicate of interest where GFP signals are co - localized with ATOH1 . Proportions of ATOH1 + ( AT + GCPs ) among electroporated . Control value is same as Fig 2G - K . Bars error bars are representing as Mean ± . Tukey - Kramer was performed between Control , S33Y S33Y + 4D ( L ) . * * * p < 0 . 001 , N . S : Not Significant . Number of : 123 , 138 113 from N = 3 mice ( S33Y ) , 144 , 223 180 from N = 3 mice ( S33Y + 4D ) .

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

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).


( A ) Coordinates for injection in normal NHPs of pilot group , study group 1 study group 2 were calculated on bases of pre - surgery MRI scans .

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

(A) 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 ) 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 midline each part subdivided in blocks . cervical spinal cord was cut in 4 blocks .

(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.


( A ) Representative confocal showing GFP + ( green ) expressing markers of neurons ( NeuN ) , astrocytes ( GFAP ) oligodendrocytes ( CNPase ) close to posterior injection site of NHP P2 . Arrows indicate co - localization of IF signals .

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

(A) 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 showing neurons ( NeuN ) overexpressing ARSA ( green ) close to posterior injection site of NHP S2 . 2 . Arrows indicate co - localization of IF signals . Note granular perinuclear ARSA , likely representing localized in Golgi / lysosomal vesicles .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 showing presence of ARSA - positive ( green ) co - stained with oligodendrocyte markers CNPase APC ( arrows ) . ( D ) Undetectable ARSA immunoreactivity in tissues of untreated . In pictures nuclei are counterstained with ToPro ( blue ) . Scale bars : A , 80 µm ; B , D 60µm , C ,

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(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


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

Vector 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).


( A ) ARSA overexpression detected by in cortical subcortical regions of injected hemispheres of NHP S2 . 3 .

(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 subcortical regions of injected hemisphere in LV . GFP - injected NHP P1 . Scale bar , 100 µm . Insets show ARSA - expressing at higher magnification . Note perinuclear accumulation of ARSA .

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(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 ) 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 Bonferroni post - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs physiological .

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(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.


( A ) Schematic representation of ARSA activity in brain of NHP S1 . 1 S2 . 2 along rostro - caudal axis ( slices 2 - 9 ) . Data are expressed as fold increase to ARSA activity detected in matched blocks of UT LV . GFP - injected NHPs ( physiological ) . Grading of colors ranges from yellow ( < 1 . 2 fold ) to green ( ≥ 2 fold ) .

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(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 ) study group 2 ( C ) ( filled grey bars , injected hemisphere ; striped grey bars , hemisphere ) , in UT LV . GFP - injected NHPs ( bars ; physiological ) . Arrows on x axis indicate injection sites . n = 2 - 12 tissue blocks / slice . Number of tissue blocks analyzed per : 51 ( UT / LV . GFP ) , 98 - 122 ( S1 . 1 S2 . 2 ) , 12 - 22 ( S1 . 2 , S1 . 3 , S2 . 2 , S2 . 3 ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs matched slices of UT / LV . GFP .

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(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 injected hemispheres of NHPs in study group 1 study group 2 . Number of tissue blocks analysed : 51 ( UT / LV . GFP ) , 105 ( study group 1 , injected hemisphere ) , 52 ( study group 1 , hemisphere ) ; 103 ( study group 2 , injected hemisphere ) , 72 ( study group 2 , hemisphere ) . One - way ANOVA Tukey ' s multiple comparison , * * * p < 0 . 001 versus UT / LV . GFP , § p < 0 . 05 versus study group 1 .

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(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 spinal cord of LV . GFP - ( n = 4 blocks / , 2 - 3 replicates ) LV . hARSA - injected NHPs ( n = 2 - 4 blocks / ; n = 10 n = 11 total blocks for study group 1 study group 2 , respectively ) . Graph on right represent summary of ARSA activity in each treatment group . One - way ANOVA Dunnet ' s multiple comparison . * p < 0 . 05 versus pilot group .

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(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 sciatic nerve of LV . GFP - ( n = 2 ) LV . hARSA - injected NHPs ( n = 3 ) . In B - F data are expressed as floating bars ( at mean ) .

(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 cerebrospinal fluid ( CSF ) of UT / LV . GFP ( n = 3 ) LV . hARSA - injected NHP ( n = 5 ) . Data are expressed as mean ± . Student t - , * p = 0 . 035 .

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(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.


( A ) Vector copy number ( VCN ) indicating distribution of integrated LV genome along rostro - caudal axis ( slices 7 - 19 ) in LV . hGALC - injected NHPs ( JT02 JV02 ) assessed by qPCR . Each dot represent VCN measured in one block within slice . Lower threshold ( dotted ) : VCN < 0 . 001 , corresponding to CT > 37 .

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(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 rostro - caudal axis of JT02 ( slices 7 - 13 ) is expressed as fold increase to region - matched blocks of hemisphere ( dotted , y = 1 ) . Data are expressed as floating bars ( min to max , at mean ; n = 4 - 8 blocks / slice ) . Arrows on x axis in A B indicate injection sites .

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(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 showing GALC + expressing markers of neurons ( NeuN ) astrocytes ( S100β ) close to injection site ( slice 10 ) in JT02 . Arrows indicate co - localization of IF signals .

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(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 showing GALC - expressing astrocytes ( S100β ; D ) neurons ( NeuN ; E ) in hemispheres , anterior ( slice 8 ) posterior ( slice 12 ) to injection site . Arrows indicate co - localization of IF signals . Scale bars : C , 70 µm ( upper panel ) 40µm ( lower panel ) ; D E , 40µm .

(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.


( A ) Specific GALC enzymatic activity in single brain slices of WT untreated ( UT ; bars , physiological ) , WT LV . hGALC - treated ( JV02 ) Krabbe - affected LV . hGALC - treated ( JV02 ) ( filled grey bars , injected hemisphere ; striped grey bars , hemisphere ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison ; * p < 0 . 05 , * * p < 0 . 01 vs matched slices of WT UT .

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(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 JV02 , as well as from WT Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis Dunn ' s multiple comparison ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT .

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(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 / ) samples from JT02 JV02 , as well as from WT Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis Dunn ' s multiple comparison ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT .

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(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 spinal cord ( SC ) tissue of untreated ( UT ) WT 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 presence ( ) or absence of 11µM AgNO3 ( ● ) .

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(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 CSF of JT02 ( pre - post - treatment ) JV02 ( post - treatment ) . Dots represent technical replicates .

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(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 , were evaluated monthly for motor performance using a modified Bayley ' s scale for infant development . For study ( JV02 JT02 ) , scores are reported as pre - surgery post - surgery . Historical data for normal affected are presented as means plus one standard deviation . Affected score significantly lower than normal at any considered . JT02 ' s motor scores increase over time post LV . GALC injection , being close to mean observed in normal juveniles at 5 months . Historical data are analyzed by two - way ANOVA Bonferroni posttests , p < 0 . 05 at 6m , p < 0 . 001 at other ages . n = 5 - 20 / group , except for normal at 6 months ( n = 1 ) .

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(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).


E . Ribosomal pull down shows lack of occupancy of ribosomes on veal2 . fli1a actb were used as positive controls .

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E. Ribosomal pull down shows lack of occupancy of ribosomes on veal2. fli1a and actb were used as positive controls.


F - I . e - GFP fusion confirms lack of peptide formation from veal2 . ( F - G ) mitfa - eGFP fused transcript . ( H - I ) veal2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected . Scale bar - 100μm .

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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.


F - T . Representative of 2 dpf zebrafish which displayed rescue of vascular integrity defects in progeny of an outcross of veal2gib005Δ8 / + zebrafish upon complementing with wild type ( WT ) veal2 . ( F - J ) Control zebrafish . ( K - O ) veal2gib005Δ8 / + injected with vehicle control . ( P - T ) veal2gib005Δ8 / + complemented with veal2 . ( F , K , P ) bright field . ( G , L , Q ) mRFP . ( H , M , R ) stained with O - dianisidine stain . ( I , N , S ) eGFP . ( J , O , T ) merged eGFP mRFP filters . Arrowheads show presence of hemorrhage due to 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 .

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F-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.


C . immunoprecipitation of veal2 was performed by pull - down of Prkcbb Epbhb3 , followed by qRT - PCR . IgG pull - down was performed as control . Gel image shows amplification of veal2 in different immunoprecipitation samples , along with 10 % input control , as marked by arrowheads .

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C. 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 of veal2 transcript across Prkcbb , Ephb3 IgG immunoprecipitations . actb was used as normalization control . Data from three different biological replicates represented as mean fold change values ± standard deviation .

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 indicated by rescue of vascular integrity defects in veal2gib005Δ8 / + . Arrowheads show presence of hemorrhage due to vascular integrity defects . Experiment was repeated in biological replicates total no of scored are mentioned on figure . ( F - I ) Magnification - 5X , Scale bar - 100μm , ( J - K ) Magnification - 20X , Scale bars - 50μm .

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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.


F . Abundance of Prkcbb in total ( T ) , ( C ) ( M ) fractions of from 2 dpf gib004Tg ( fli1a : EGFP ; gata1a : DsRed ) veal2gib005Δ8 / + zebrafish . Arrowhead indicates Prkcbb enrichment in fraction of veal2gib005Δ8 / + zebrafish .

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F. 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.


E - H e - GFP fusion confirms lack of peptide formation from veal2 . ( E - F ) mitfa - eGFP fused transcript . ( G - H ) VEAL2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected . Scale bar -

E-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-100μ


I - N . Complementation of VEAL2 in veal2gib005Δ8 / + significantly rescued hemorrhage phenotype . Arrowheads show presence of hemorrhage due to vascular integrity defects . ( I - L ) Magnification - 5X , Scale bar - 100μm ( M - N ) Magnification - 20X , Scale bars - 50μm .

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I-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 smFISH of VEAL2 highlight their colocalization . ( R ) PRKCB in GFP channel . ( S ) VEAL2 in CAL - fluor red ( 610nM ) . ( T ) Merged image for PRKCB VEAL2 . Magnification - 100X , scale bar - 20μm .

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.


K - AB . VEAL2 regulates junctional dynamics by interacting with PRKCB . Overexpression of VEAL2 retains PRKCB mostly in cytoplasm keeps strong junctional assembly formation of CDH5 CTNNB1 on . Knockdown of VEAL2 led to migration of PRKCB on henceforth degradation of junctional assembly of CDH5 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 in HUVECs . ( W - AB ) Dot plot representing of signal localization in / total fraction . was done using imageJ . Data from of different fields of 3 technical replicates of 1 biological replicate is presented as representation . Data is shown as values ; middle bar represents mean error bar represents ± standard deviation .

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K-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.


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

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E-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.


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

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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


A - C Exponentially growing cultures ( exp ) were arrested in G1 with α - factor ( αf ) released into YEPD with or without CPT ( 50 μM ) . samples were harvested at indicated time points to evaluat Rad53 phosphorylation by western blot with anti - Rad53 antibodies ( C )

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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 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 with anti - Rad53 antibodies

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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


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

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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


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

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(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 cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at indicated time points were subjected to western blot with anti - Rad53 antibodies ( B , D

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(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


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

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


HeLa cyclin F Knock - Out ( CCNF K / O ) 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 ( right ) .

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HeLa 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).


survival measured using rezasurin compared to controls treated with DMSO ( expressed as relative proliferation % ) . were treated with indicated inhibitors at 0 . 1 1 μM .

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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.


survival measured using rezasurin compared to controls ( 0 ) treated with DMSO ( expressed as relative proliferation % ) . were treated with UCN - 01 at indicated concentrations .

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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 CCNF K / O after treatment with specific Chk1i ( LY2603618 ) at indicated concentrations plotted on a log10 scale to measure differences in IC50 .

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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 was measured in HeLa CCNF K / O left untreated or treated with Chk1i ( LY2603618 ) for 24h , 48h , or 72h as indicated . ( n . s . = non - significant ) .

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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).


viability measurements using Propidium Iodide for HeLa CCNF K / O after treatment with Chk1i ( LY2603618 ) .

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Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K/O cells after treatment with Chk1i (LY2603618).


U - 2 - OS were seeded transfected with non - targeting siRNA siNC ( Negative Control ) or siCyc F . 24 hours after transfection , were treated with UCN - 01 , or Chk1i ( LY2603618 ) at indicated concentrations for 3 days before viability was measured .

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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.


viability measurements using Propidium Iodide in U - 2 - OS transfected with non - targeting siNC siCyc F treated with Chk1i ( LY2603618 ) as indicated .

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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.


treated with 1μM Chk1i ( LY2603618 ) for indicated hours ( h ) were harvested lysed using SDS . Indicated were resolved by SDS page detected by Western blot ( WB ) . TFIIH was used as a loading control .

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Cells 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.


( % ) of γ - H2AX positive in G1 after Chk1i ( LY2603618 ) treatment for indicated hours ( h ) measured using Activated Sorting ( FACS ) . G1 were considered having DAPI 2n EdU negative . ( % ) of γ - H2AX positive in replicating ( EdU + ) after Chk1i ( LY2603618 ) treatment at indicated time points . ( % ) of γ - H2AX positive in G2 / M after Chk1i ( LY2603618 ) treatment . G2 / M were considered having DAPI 4n EdU negativity .

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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.


of γ - H2AX measured as Relative Units ( RFU ) in early S phase treated with Chk1i . Mean of γ - H2AX was evaluated from EdU + with 2N DAPI content . of γ - H2AX measured as Relative Units ( RFU ) in replicating treated with Chk1i . Mean of γ - H2AX was evaluated from EdU + . Overall , cycle profile of HeLa CCNF K / O after Chk1i treatment . with 2n DAPI but EdU - were gated as G1 ; with > 2n but < 4n DAPI but EdU - were gated as EdU - S ; with > 2n but < 4n DAPI but EdU + were gated as EdU + S phase ; with 4n DAPI pH3 S10 negative were gated as G2 phase with 4n DAPI pH3 S10 positive as M . Data from at least 3 independent replicates plotted as Mean ± SD .

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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 CCNF K / O after treatment with 1μM Chk1i ( LY2603618 ) at indicated hours ( h ) . Blue labeled dots represent γ - H2AX positive . Relates to materials section for details on representation .

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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.


Representative plots of γ - H2AX vs DAPI signal of CCNF K / O transfected with empty GFP GFP - CCNF untreated ( top panels ) after treatment with 1μM Chk1i ( LY2603618 ) for 20h ( bottom panels ) . Normalised γ - H2AX in CCNF K / O transfected with either GFP empty vector or GFP - Cyc F treated with 1μM Chk1i for 20 hours . GFP positive were gated . Data from 3 independent experiments were plotted with Mean % ± SD .

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Representative 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 of neutral alkaline comet from HeLa CCNF K / O treated with Chk1i . were treated with 1μM Chk1i for 24h before being harvested for neutral alkaline comet assays as indicated . of comets in alkaline gels . At least 100 , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using Mann - Whitney ( two - ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % / 100 . of comets in neutral gels . At least 100 , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using Mann - Whitney ( two - ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % / 100 .

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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.


( % ) of γ - H2AX positive in HeLa CCNF K / O after transfection of indicated siRNAs treated with Chk1i for 20 hours . of γ - H2Ax positive were plotted as Mean % ± SD . ( % ) of hyper - RPA positive in HeLa CCNF K / O after transfection of indicated siRNAs treated with Chk1i for 20 hours . of hyper RPA positive were plotted as Mean % ± SD .

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Percentage (%) of γ-H2AX positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of γ-H2Ax positive cells were plotted as Mean % ± SD. Percentage (%) of hyper-RPA positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of hyper RPA positive cells were plotted as Mean % ± SD.


( % ) of γ - H2AX positive in S phase by DAPI content after Chk1i ( LY2603618 )

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Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618)


Relative γ - H2AX in transfected with either Empty Vector ( EV ) or HA - E2F1 treated with Chk1i for 20 hours . γ - H2AX in HA positive was normalized to empty vector transfected compared to not expressing HA .

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Relative γ-H2AX intensity in cells transfected with either Empty Vector (EV) or HA-E2F1 treated with Chk1i for 20 hours. γ-H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA.


HeLa CCNF K / O transfected with indicated siRNA treated with 1μM Chk1i ( LY2603618 ) for 20 hours were harvested lysed using SDS . Indicated were resolved by SDS page detected by WB . TFIIH served as a loading control .

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HeLa and CCNF K/O cells transfected with the indicated siRNA and treated with 1μM Chk1i (LY2603618) for 20 hours were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. TFIIH served as a loading control.


HeLa CCNF K / O were harvested lysed using SDS . Indicated were resolved by SDS page detected by WB . Ponceau S was used as a loading control .

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HeLa and CCNF K/O cells were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. Ponceau S staining was used as a loading control.


U - 2 - OS transfected with non - targeting siRNA ( siNC ) or with an siRNA targeting Cyc F were lysed using SDS . Whole lysates were analyzed by immunoblotting 48h after transfection . H3 was used as a loading control .

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U-2-OS cells transfected with non-targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS. Whole cell lysates were analyzed by immunoblotting 48h after transfection. H3 was used as a loading control.


showing gene set variance ( GSVA ) score for gene sets related to E2F targets ( PID _ E2F _ PATHWAY , ISHIDA _ E2F _ TARGETS HALLMARK _ E2F _ TARGETS ) .

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Heatmap showing gene set variance analysis (GSVA) score for gene sets related to E2F targets (PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS).


HEK293T transfected with empty vector ( EV ) , FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) FLAG - cyclin F M309A were treated with MLN4924 for 4 hours harvested for immunoprecipitation using FLAG antibodies . Immunoprecipitates were immunoblotted as indicated .

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HEK293T cells transfected with empty vector (EV), FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated.


HEK293T were cotransfected with MYC - tagged ubiquitin HA - E2F1 in presence of FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) FLAG - cyclin F M309A were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions immunoblotted .

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HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 in the presence of FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HeLa CCNF K / O treated with 10 μM Chk1i ( LY2603618 ) for indicated time points ( h = hours ) were harvested lysed using SDS . Indicated were resolved by SDS page detected by WB . H2B served as a loading control .

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HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h=hours) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. H2B served as a loading control.


HEK293 transfected with empty vector ( EV ) or HA - E2F1 were treated with Chk1i for 20 hours harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated .

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HEK293 cells transfected with empty vector (EV) or HA-E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.


of immunoprecipitation results of Figure 5G from three independent experiments . Data from 3 independent experiments were plotted with Mean % ± SD . * < 0 . 05 p - value calculated by two - paired t - .

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Quantification of immunoprecipitation results of Figure 5G from three independent experiments. Data from 3 independent experiments were plotted with Mean % ± SD. *< 0.05 p-value and calculated by two-tailed paired t-test.


HEK293T were cotransfected with MYC - tagged ubiquitin HA - E2F1 were treated with Chk1i for 20 hours siRNA of cyclin F were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions immunoblotted .

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HEK293T transfected with HA - E2F1 Wild Type ( WT ) HA - E2F1 lacking a CY motif ( ∆ RxL ) harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated .

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

HEK293T cells transfected with HA-E2F1 Wild Type (WT) and HA-E2F1 lacking a CY motif (∆RxL) harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.


HEK293T were cotransfected with MYC - tagged ubiquitin HA - E2F1 or HA - E2F1 ∆ RxL lacking CY motif as indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions immunoblotted .

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 or HA-E2F1 ∆RxL lacking the CY motif as indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HeLa stably expressing HA - E2F1 WT HA - E2F1 ∆ RxL under control of a retroviral promoter were treated with Cycloheximide ( CHX ) at 50 μg / ml for indicated minutes ( min ) of E2F1 half -

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

HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min) Quantification of E2F1 half-life


HeLa stably expressing an Empty Vector ( EV ) , HA - E2F1 WT HA - E2F1 ∆ RxL under control of a retroviral promoter were treated with Chk1i for indicated hours ( h ) . Indicated were resolved by SDS page detected by WB . H3 was used as a loading control .

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

HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours (h). Indicated protein were resolved by SDS page and detected by WB. H3 was used as a loading control.


Relative survival ( % ) of HeLa stably expressing an Empty Vector ( EV ) , HA - E2F1 WT HA - E2F1 ∆ RxL under control of a retroviral promoter measured using rezasurin compared to EV treated with Chk1i at indicated concentrations .

Relative survival (%) of HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations.


A . Representative plots of splenocytes from CD45 . 1 mice adoptively transferred with CD45 . 2 OT - II WT or CCR5 - / - lymph node suspensions , five after infection with rVACV - OVA . gating strategy used to identify memory CD4 + T subtypes is shown ( n = 5 ) .

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

A. Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT-II WT or CCR5-/- lymph node cell suspensions, five weeks after infection with rVACV-OVA virus. The gating strategy used to identify the memory CD4+ T cell subtypes is shown (n = 5).


B . Absolute number of OT - II recovered in spleens of mice as in A ( n = 5 ) .

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

B. Absolute number of OT-II cells recovered in spleens of mice as in A (n = 5).


C , of CD4 + ( C ) in OT - II WT CCR5 - / - populations ( n = 5 ) .

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

C, Percentage of CD4+ TEM (C) in the OT-II WT and CCR5-/- populations (n = 5).


D . of TCM ( D ) in OT - II WT CCR5 - / - populations ( n = 5 ) .

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

D. Percentage of TCM (D) in the OT-II WT and CCR5-/- populations (n = 5).


E . IFNγ - producing OT - II WT CCR5 - / - memory isolated from mice as in A restimulated ex vivo with OVA323 - 339 ( 1 μM ) ( n = 4 ) .

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

E. IFNγ-producing OT-II WT and CCR5-/- memory cells isolated from mice as in A and restimulated ex vivo with OVA323-339 (1 μM) (n = 4).


G - I . Representative plots ( G ) of frequency ( H ) absolute number ( I ) of Tfh ( CD4 + CD44 + PD - 1 + CXCR5 + ) in spleen after immunization ( day 7 ) with NIP - OVA ( n = 7 ) .

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

G-I. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of Tfh cells (CD4+CD44+PD-1+CXCR5+) in the spleen after primary immunization (day 7) with NIP-OVA (n = 7).


J , K . ELISA of high - ( J ) low - affinity ( K ) isotype - specific anti - NIP antibodies in sera from OVA / OVA - OVA / KLH - immunized mice ( day 15 post - challenge ; n = 5 mice / group ) . Data representative of one experiment of two .

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

J, K. ELISA analysis of high- (J) and low-affinity (K) isotype-specific anti-NIP antibodies in sera from OVA/OVA- and OVA/KLH-immunized mice (day 15 post-challenge; n = 5 mice/group). Data representative of one experiment of two.


A , Representative histograms of mean ( MFI ; A ) in OT - II WT CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± ( n ≥ 3 ) .

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

A, Representative histograms and quantification of mean fluorescence intensity (MFI; A) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).


B . of positive for indicated memory markers ( B ) in OT - II WT CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± ( n ≥ 3 ) .

B. the percentage of cells positive for the indicated memory markers (B) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).


C - F . IL - 2 - ( C , D ) IL - 15 - expanded lymphoblasts ( E , F ) were restimulated with indicated concentrations of OVA323 - 339 ; proliferation ( thymidine incorporation into ; C , E ) IL - 2 production ( by ELISA ; D , F ) were measured after 72 h . Data are presented as mean ± ( n = 5 ) .

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

C-F. IL-2- (C, D) and IL-15-expanded lymphoblasts (E, F) were restimulated with indicated concentrations of OVA323-339; cell proliferation (thymidine incorporation into DNA; C, E) and IL-2 production (by ELISA; D, F) were measured after 72 h. Data are presented as mean ± SEM (n = 5).


A - C . of TCR nanoclustering by EM in OT - II WT CCR5 - / - naïve ( A ; n = 6 / genotype ; WT : 3427 , CCR5 - / - : 3528 particles ) , IL - 2 - ( B ; WT , n = 8 , 15419 particles ; CCR5 - / - , n = 6 , 5410 particles ) or IL - 15 - expanded lymphoblasts ( C ; WT , n = 8 , 27518 particles ; CCR5 - / - , n = 7 , 22696 particles ) . A representative small field image at top of each panel shows gold particle distribution in surface replicas of anti - CD3ε - labeled ; at bottom , ( mean ± ) of gold particles in clusters of indicated in WT ( gray bars ) CCR5 - / - ( red ) . Insets show distribution of clusters of one , two , three , four , or more than four particles , statistical .

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

A-C. Analysis of TCR nanoclustering by EM in OT-II WT and CCR5-/- naïve cells (A; n = 6 cells/genotype; WT: 3427, CCR5-/-: 3528 particles), and IL-2- (B; WT, n = 8 cells, 15419 particles; CCR5-/-, n = 6 cells, 5410 particles) or IL-15-expanded lymphoblasts (C; WT, n = 8 cells, 27518 particles; CCR5-/-, n = 7 cells, 22696 particles). A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti-CD3ε-labeled cells; at bottom, quantification (mean ± SEM) of gold particles in clusters of indicated size in WT (gray bars) and CCR5-/- cells (red). Insets show the distribution of clusters of one, two, three, four, or more than four particles, and statistical analysis.


D , E . Posterior distribution in naïve ( D ) IL - 2 - expanded lymphoblasts ( E ) of clustering parameter b for WT ( gray ) CCR5 - / - ( red ) ; randomly generated distributions of receptors are shown in blue . mean value of b parameter is indicated for each condition . probability of a chance distribution similar to that determined in is nearly 0 % by ROPE .

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

D, E. Posterior distribution in naïve (D) and IL-2-expanded lymphoblasts (E) of the clustering parameter b for WT (gray) and CCR5-/- cells (red); randomly generated distributions of receptors are shown in blue. The mean value of the b parameter is indicated for each condition. The probability of a chance distribution similar to that determined in cells is nearly 0% by the ROPE.


F . Comparison of TCR oligomer using BN - PAGE anti - CD3ζ immunoblotting in day 10 , IL - 2 - expanded WT CCR5 - / - OT - II lymphoblasts lysed in buffer containing digitonin or Brij - 96 . marker is ferritin ( f1 , 440 f2 , 880 kDa forms ) . of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry ( right ; n = 5 ) .

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

F. Comparison of TCR oligomer size using BN-PAGE and anti-CD3ζ immunoblotting in day 10, IL-2-expanded WT and CCR5-/- OT-II lymphoblasts lysed in buffer containing digitonin or Brij-96. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry (right; n = 5).


G . Top , representative small field EM showing gold particle distribution in surface replicas of CD4 + T isolated from OVA / OVA - immunized WT CCR5 - / - mice . Bottom , ( mean ± ) of gold particles in clusters of indicated ( WT , gray bars ; n = 5 , 14680 particles ; CCR5 - / - , red ; n = 7 , 15374 particles ) . Insets show distribution between clusters of one , two , three , four or more than four particles , statistical .

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

G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5-/- mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14680 particles; CCR5-/-, red; n = 7 cells, 15374 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


A . OT - II WT were activated with OVA323 - 339 , alone or with TAK - 779 . After three days , antigen TAK - 779 were removed lymphoblasts expanded in IL - 2 - containing medium . TCR nanoclustering was analyzed in anti - CD3ε - labeled surface replicas of day 10 lymphoblasts . Top , representative small field EM showing gold particle distribution in surface replicas of WT CD4 + T alone or with TAK - 779 . Bottom , of gold particles in clusters of indicated . Inset , distribution of gold particles between clusters of one , two , three , four or more than four particles in vehicle - ( gray bars ; n = 5 , 11266 particles ) TAK - 779 - treated ( black ; n = 6 , 5138 particles ) . B . OT - II WT were activated with OVA323 - 339 , TAK - 779 was added at days 3 , 5 , 7 after antigen removal . as above , untreated ( gray bars ; n = 5 , 6400 particles ) TAK - 779 - treated ( black ; n = 6 , 7153 particles ) . Inset show distribution between clusters of one , two , three , four or more than four particles , statistical .

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

A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles). B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


C . OT - II WT naïve were activated with antigen in presence or not of CXCR4 inhibitor AMD3100 . Left , representative EM showing gold particle distribution in surface replicas . Right , of gold particles in clusters as above , vehicle - ( gray bars ; n = 6 , 12339 particles ) AMD3100 - treated ( black ; n = 7 , 17059 particles ) . Inset show distribution between clusters of one , two , three , four or more than four particles , statistical .

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

C. OT-II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100. Left, representative EM images showing gold particle distribution in the cell surface replicas. Right, analysis of gold particles in clusters as above, vehicle- (gray bars; n = 6 cells, 12339 particles) and AMD3100-treated cells (black; n = 7 cells, 17059 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


A . Total Chol in WT CCR5 - / - OT - II lymphoblasts ( day 10 , IL - 2 - expanded ) as determined by a fluorometric ( n = 6 ) .

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

A. Total Chol levels in WT and CCR5-/- OT-II lymphoblasts (day 10, IL-2-expanded) as determined by a fluorometric assay (n = 6).


B - D . SM ( B ) , Cer ( C ) dhCer ( D ) in WT CCR5 - / - OT - II 10 - day lymphoblasts , as determined by UPLC - TOF MS . Values , after normalization with C17 standards number in each sample , are mean of two independent experiments ( n = 6 ) .

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

B-D. SM (B), Cer (C) and dhCer (D) levels in WT and CCR5-/- OT-II 10-day lymphoblasts, as determined by UPLC-TOF MS. Values, after normalization with C17 standards and cell number in each sample, are the mean of two independent experiments (n = 6).


E . RT - qPCR determination of CerS mRNA in naïve IL - 2 - expanded WT CCR5 ‑ / - OT - II 10 - day lymphoblasts ( n = 3 - 5 ) .

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

E. RT-qPCR determination of CerS mRNA levels in naïve and IL-2-expanded WT and CCR5‑/- OT-II 10-day lymphoblasts (n = 3-5).


F . Representative immunoblot showing CerS2 in naïve WT CCR5 - / - OT - II 10 - day lymphoblasts , densitometric of blots as above ( n = 10 ) .

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

F. Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5-/- OT-II 10-day lymphoblasts, and densitometric quantification of blots as above (n = 10).


G . ChIP of CerS2 promoter using an anti - H3K9Ac antibody . Scheme of CerS2 promoter showing CpG islands primers used for amplification . Relative ChIP of CerS2 promoter in WT CCR5 - / - OT - II 10 - day lymphoblasts ( n = 3 ) .

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

G. ChIP analysis of the CerS2 promoter using an anti-H3K9Ac antibody. Scheme of the CerS2 promoter showing CpG islands and primers used for amplification. Relative ChIP of the CerS2 promoter in WT and CCR5-/- OT-II 10-day lymphoblasts (n = 3).


H . Relative CerS2 mRNA level in CD4 T treated with PTx ( n = 3 ) .

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

H. Relative CerS2 mRNA level in CD4 T cells treated with PTx (n = 3).


L . Representative immunofluorescence showing pSer142 - GATA - 1 ( green ) of OT - II WT CCR5 - / - lymphoblasts . green channel ( top ) merge with DAPI ( blue ; bottom ) are shown . Scale bar , 10 μm . M . of of plotted as integrated density in DAPI - stained ; n ≥ 50 / condition ) .

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

L. Representative immunofluorescence images showing pSer142-GATA-1 staining (green) of OT-II WT and CCR5-/- lymphoblasts. The green channel (top) and the merge with nuclear DAPI staining (blue; bottom) are shown. Scale bar, 10 μm. M. Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI-stained area; n ≥50 cells/condition).


N . Top , basic scheme of CerS2 promoter , indicating putative GATA - 1 binding site ( blue ) location of primers used for amplification in ChIP assays ( black arrows ) . Bottom , relative anti - GATA - 1 ChIP in OT - II WT CCR5 - / - lymphoblasts ( n = 5 ) .

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

N. Top, basic scheme of the CerS2 promoter, indicating the putative GATA-1 binding site (blue) and location of the primers used for amplification in ChIP assays (black arrows). Bottom, relative anti-GATA-1 ChIP levels in OT-II WT and CCR5-/- lymphoblasts (n = 5).


A . Scheme of strategy used to form TCR proteoliposomes , of LUV generated at indicated lipid molar . Polydispersity index values are shown as black squares for each condition ( n = 3 ) . SBP , streptavidin - binding - peptide - tagged TCR .

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

A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition (n = 3). SBP, streptavidin-binding-peptide-tagged TCR.


B . Representative immunoblots comparing TCR nanocluster sizes via BN - PAGE anti - CD3ζ immunoblotting in TCR proteoliposomes lysed in presence of Brij - 96 or digitonin . marker is ferritin ( f1 , 440 f2 , 880 kDa forms ) .

O O O O O O O O O O O O O O O O O O O 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. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms).


C . of nanocluster monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B ( n ≥ 4 ) .

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

C. The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B (n ≥ 4).


D . Cer in OT ‑ II 10 - day lymphoblasts , untreated or treated with SMase ( n = 4 ) .

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

D. Cer levels in OT‑II 10-day lymphoblasts, untreated or treated with SMase (n = 4).


E . Representative small field showing gold particle distribution , ( mean ± ) of gold particles in clusters of indicated in surface replicas from untreated ( gray bars ; n = 5 , 8126 particles ) SMase - treated ( 1h ) OT - II lymphoblasts ( black cyan ; n = 6 , 8457 particles ) after CD3ε labeling , as determined by EM . inset shows distribution between clusters of one , two , three , four or more than four particles .

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

E. Representative small field images showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black cyan; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles.


F . GFP expression in shCtrl - ( black ) shCerS2 ( orange ) - transduced 2B4 after puromycin selection , as determined by FACS .

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

F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS.


G . Relative CerS2 mRNA in TAK - 779 - treated 2B4 as in F . Values were normalized to those obtained in untransduced TAK - 779 - treated 2B4 ( n = 3 ) .

G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F. Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells (n = 3).


H . Representative immunoblot with anti - CerS2 antibody to determine CerS2 in shCtrl ShCerS2 - transduced 2B4 as in G . Filters were rehybridized with β - actin as loading control .

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

H. Representative immunoblot with anti-CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2-transduced 2B4 cells as in G. Filters were rehybridized with β-actin as loading control.