( B ) Effect of lipin - 1γ overexpression on the metabolic fate of de novo synthesized PA . Experiments were performed as in panel A , except that the membranes were also prepared from cells expressing a GFP - tagged lipin 1γ , as indicated . Note the large amount of TG and increased production of PC under these conditions . Results of a representative experiment are shown , and these experiments were repeated with identical results .

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

(B) Effect of lipin-1γ overexpression on the metabolic fate of de novo synthesized PA. Experiments were performed as in panel A, except that the membranes were also prepared from cells expressing a GFP-tagged lipin 1γ, as indicated. Note the large amount of TG and increased production of PC under these conditions. Results of a representative experiment are shown, and these experiments were repeated with identical results.


Effect of different fatty - acyl - CoA analogs ( each used in 10 µM ) on the metabolic fate of de novo synthesized PA . Note the preferential conversion of oleoyl - CoA containing PA into TG and the very poor incorporation of stearoyl - CoA into TG . Arachidonoyl - CoA is a relatively poor substrate for the generation of de novo synthesized PA ; however , this substrate has the highest conversion rate into PI , which was calculated as the ratio of the PI and PA mass shown in panel D . , while quantified values ( means ± range ) from two such experiments are shown in ( D ) and ( E ) .

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

Effect of different fatty-acyl-CoA analogs (each used in 10 µM) on the metabolic fate of de novo synthesized PA. Note the preferential conversion of oleoyl-CoA containing PA into TG and the very poor incorporation of stearoyl-CoA into TG. Arachidonoyl-CoA is a relatively poor substrate for the generation of de novo synthesized PA; however, this substrate has the highest conversion rate into PI, which was calculated as the ratio of the PI and PA mass shown in panel D. , while quantified values (means ± range) from two such experiments are shown in (D) and (E).


( E and F ) RT - PCR and western blot showing the effect of RNAi - mediated B52 depletion in S2R + cells ( E ) and in larval wing discs ( F ) .

O O O O O O O O O O O O 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 and F) RT-PCR and western blot showing the effect of RNAi-mediated B52 depletion in S2R+ cells (E) and in larval wing discs (F).


( D ) Quantification of Posterior / Total wing area in male flies of the genotypes illustrated in panel C . UAS - GFP was used as a control . Each point represents a single wing , bars represent mean with standard deviation . * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) . Flies were grown at 18 ° C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of Posterior/Total wing area in male flies of the genotypes illustrated in panel C. UAS-GFP was used as a control. Each point represents a single wing, bars represent mean with standard deviation. *** p-value<0.001, **** p-value<0.0001 (unpaired two-tailed t-tests). Flies were grown at 18°C.


( C ) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain ( hh - Gal4 driver ) . For each target gene , relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti - ßgal antibody ( for ex - lacZ and diap1 - lacZ ) or direct visualization of GFP ( ban - sensor , in this case posterior domain was visualized by immunostaining against V5 - tag present in UAS - Yki transgenes ) . In the scatter dot plot , each symbol ( circle , square and triangle ) represents a single wing disc , bars represent mean with SEM . * * p - value < 0 . 01 ; * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain (hh-Gal4 driver). For each target gene, relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti-ßgal antibody (for ex-lacZ and diap1-lacZ) or direct visualization of GFP (ban-sensor, in this case posterior domain was visualized by immunostaining against V5-tag present in UAS-Yki transgenes). In the scatter dot plot, each symbol (circle, square and triangle) represents a single wing disc, bars represent mean with SEM. ** p-value<0.01; **** p-value<0.0001 (unpaired two-tailed t-tests).


( C ) Comparison of Yki isoforms binding capabilities by dual - luciferase co - IP . Graphs show the normalized co - IP ( NcoIP ) results for 13 proteins tested against the two Yki isoforms and the negative control ( Flag - Firefly ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of Yki isoforms binding capabilities by dual-luciferase co-IP. Graphs show the normalized co-IP (NcoIP) results for 13 proteins tested against the two Yki isoforms and the negative control (Flag-Firefly).


( D ) Interaction between Sd and Tgi in absence or presence of Yki isoforms . ( E ) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms . ( F ) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms . In C , D , E and F , bars represent mean and error bars represent SD , n = 4 . p - values : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( unpaired two - tailed t - tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Interaction between Sd and Tgi in absence or presence of Yki isoforms. (E) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms. (F) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms. In C, D, E and F, bars represent mean and error bars represent SD, n=4. p-values: *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001 (unpaired two-tailed t-tests).


( G ) Luciferase assay using 3xSd - Luciferase reporter in S2 cells , transfected with single or combination of Yki isoforms . Error bars represent SD , n = 6 . * p - value < 0 . 05 ; * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase assay using 3xSd-Luciferase reporter in S2 cells, transfected with single or combination of Yki isoforms. Error bars represent SD, n=6. * p-value<0.05; **** p-value<0.0001 (unpaired two-tailed t-tests).


( H ) Co - overexpression of Yki isoforms using GMR - Gal4 driver provides evidence of competition between Yki isoforms .

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

(H) Co-overexpression of Yki isoforms using GMR-Gal4 driver provides evidence of competition between Yki isoforms.


( C ) Comparison of posterior / total area ratio of females wings upon depletion of B52 in a yki + ( wild type ) or yki2 - only background . Each point corresponds to a single wing , bars represent mean with SD ( unpaired two - tailed t - tests : * * * * p - value < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of posterior/total area ratio of females wings upon depletion of B52 in a yki+ (wild type) or yki2-only background. Each point corresponds to a single wing, bars represent mean with SD (unpaired two-tailed t-tests: **** p-value <0.0001).


( E and F ) Quantification of sibling clones size in the wing pouch . In ( D ) , sibling wild - type clones are labelled with 2xGFP and 2xRFP . In ( E ) , unlabelled clones are homozygous for the yki2 - only - A allele whereas sibling clones ( RFP / RFP ) are wild type . In both graphs , clones pairs are sorted according to RFP / RFP clones size .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) Quantification of sibling clones size in the wing pouch. In (D), sibling wild-type clones are labelled with 2xGFP and 2xRFP. In (E), unlabelled clones are homozygous for the yki2-only-A allele whereas sibling clones (RFP/RFP) are wild type. In both graphs, clones pairs are sorted according to RFP/RFP clones size.


( C ) Distribution of wing size and wing asymmetry among the flies analyzed in panel B . Each circle represents the area of a single wing . Each wing was measured in duplicate . The difference between Right ( orange ) and Left ( blue ) sides of a single fly is schematized by a black line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of wing size and wing asymmetry among the flies analyzed in panel B. Each circle represents the area of a single wing. Each wing was measured in duplicate. The difference between Right (orange) and Left (blue) sides of a single fly is schematized by a black line.


C . Representative western blot showing changes in various ER stress markers and O - GlcNAcylation - related protein expression in THP - 1 cells treated with Stx2a ( 10 ng / mL ) for 3 h in the presence or absence of O - linked N - acetylglucosamine transferase ( OGT ) inhibitor OSMI - 1 ( 10 µM final ) or serine / threonine - protein kinase / endoribonuclease inositol - requiring enzyme 1 a ( IRE1a ) inhibitor MKC - 3946 ( 10 µM final ) . D . Quantification of the band intensities in ( C ) . Data are presented as mean ± S . E . M . ( n = 3 biological replicates ) normalized against β - actin , which was used as a loading control . Data information : Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

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

C. Representative western blot showing changes in various ER stress markers and O-GlcNAcylation-related protein expression in THP-1 cells treated with Stx2a (10 ng/mL) for 3 h in the presence or absence of O-linked N-acetylglucosamine transferase (OGT) inhibitor OSMI-1 (10 µM final) or serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 a (IRE1a) inhibitor MKC-3946 (10 µM final). D. Quantification of the band intensities in (C). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


E . Representative western blot of O - GlcNAcylation and pro - caspase - 3 cleavage in THP - 1 cells treated with Stx2a ( 10 ng / mL ) in the presence or absence of OGT inhibitor OSMI - 1 ( 10 µM final ) . F . Quantification of the band intensities for O - GlcNAcylation ( RL - 2 ) in ( E ) . Data are presented as mean ± S . E . M . ( n = 3 biological replicates ) normalized against β - actin , which was used as a loading control . The effects of Stx2a - mediated induction for O - GlcNAc levels at each time - point were compared to 0 h ( left panel ) , and OSMI - 1 treatment was compared with that of the vehicle ( DMSO ) control at each time - point ( right panel ) . Data information : Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

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

E. Representative western blot of O-GlcNAcylation and pro-caspase-3 cleavage in THP-1 cells treated with Stx2a (10 ng/mL) in the presence or absence of OGT inhibitor OSMI-1 (10 µM final). F. Quantification of the band intensities for O-GlcNAcylation (RL-2) in (E). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. The effects of Stx2a-mediated induction for O-GlcNAc levels at each time-point were compared to 0 h (left panel), and OSMI-1 treatment was compared with that of the vehicle (DMSO) control at each time-point (right panel). Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


B . ELISAs were used to analyze the inhibitory effect of OSMI - 1 ( 10 µM , final ) on cytokine and chemokine production from THP - 1 cells exposed to Stx2a ( 10 ng / mL ) for 9 h ( n = 3 biological replicates ) . Data information : For graphs error bar represents mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . The effects of OSMI - 1 were compared with those of the vehicle ( DMSO ) control at each time - point .

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

B. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on cytokine and chemokine production from THP-1 cells exposed to Stx2a (10 ng/mL) for 9 h (n = 3 biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.


C . Real - time qRT - PCR analyses of fold changes in transcript levels for each cytokine / chemokine gene in D - THP - 1 cells following Stx2a ( 10 ng / mL ) intoxication for various time - points in the presence or absence of OSMI - 1 ( 10 µM , final ) . Data are normalized using GAPDH as a loading control ( n = 3 , biological replicates ) . Data information : For graphs error bar represents mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . The effects of OSMI - 1 were compared with those of the vehicle ( DMSO ) control at each time - point .

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

C. Real-time qRT-PCR analyses of fold changes in transcript levels for each cytokine/chemokine gene in D-THP-1 cells following Stx2a (10 ng/mL) intoxication for various time-points in the presence or absence of OSMI-1 (10 µM, final). Data are normalized using GAPDH as a loading control (n = 3, biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.


A . Representative western blot showing changes of overall O - GlcNAcylation ( upper panel ) or phosphorylation status of each target protein ( lower panels ) in THP - 1 cells treated with Stx2a ( 10 ng / mL ) for 9 h in the presence or absence of OSMI - 1 ( 10 µM , final ) . B . Quantification of the relative band intensities for O - GlcNAcylation ( RL - 2 ) or each of the phosphorylated - and total - protein bands in ( A ) . Data are normalized against β - actin , which was used as a loading control ( n = 3 biological replicates ) . The effects of Stx2a in vehicle controls were compared with lysates prepared from cells maintained in the absence of Stx2a , and OSMI - 1 treatment was compared with that of the vehicle ( DMSO ) controls in the presence of Stx2a . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O 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 O O O O O O O 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 western blot showing changes of overall O-GlcNAcylation (upper panel) or phosphorylation status of each target protein (lower panels) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of OSMI-1 (10 µM, final). B. Quantification of the relative band intensities for O-GlcNAcylation (RL-2) or each of the phosphorylated- and total-protein bands in (A). Data are normalized against β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with lysates prepared from cells maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C . Representative western blot showing changes in OGT expression ( top panel ) , overall O - GlcNAcylation ( second panel ) , or phosphorylation status of each target protein ( lower panels ) in THP - 1 cells treated with Stx2a ( 10 ng / mL ) for 9 h in the presence or absence of OGT knockdown . D . Quantification of the relative band intensities between each of phosphorylated - and total - protein bands in ( C ) . Data are normalized against β - actin , which was used as a loading control ( n = 3 biological replicates ) . The effects of Stx2a in siScr controls were compared with those of lysates from cells maintained in the absence of Stx2a , and OGT knockdown was compared with that of siScr controls in the presence of Stx2a . ( n . s . = not significant ) . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

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

C. Representative western blot showing changes in OGT expression (top panel), overall O-GlcNAcylation (second panel), or phosphorylation status of each target protein (lower panels) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of OGT knockdown. D. Quantification of the relative band intensities between each of phosphorylated- and total-protein bands in (C). Data are normalized against β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a in siScr controls were compared with those of lysates from cells maintained in the absence of Stx2a, and OGT knockdown was compared with that of siScr controls in the presence of Stx2a. (n.s. = not significant). Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


G . Representative western blot showing changes of overall O - GlcNAcylation ( upper panel ) , phosphorylation status of each target protein ( middle panels ) or pro - caspase - 3 cleavage ( lower panel ) in THP - 1 cells treated with Stx2a ( 10 ng / mL ) for 9 h in the presence or absence of overexpression of Akt ( wild - type or T305A / T312A ) . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative western blot showing changes of overall O-GlcNAcylation (upper panel), phosphorylation status of each target protein (middle panels) or pro-caspase-3 cleavage (lower panel) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of overexpression of Akt (wild-type or T305A/T312A). Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


A . Representative western blot showing the changes of O - GlcNAcylation in primary human renal proximal tubule epithelial ( HRPTEpi ) cells ( originating from fetal or adult kidney ) treated with different concentrations ( 0 - 10 ng / mL ) of Stx2a for 24 h .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 western blot showing the changes of O-GlcNAcylation in primary human renal proximal tubule epithelial (HRPTEpi) cells (originating from fetal or adult kidney) treated with different concentrations (0-10 ng/mL) of Stx2a for 24 h.


B . LDH cytotoxicity assay following Stx2a ( 10 ng / mL ) treatment of HRPTEpi ( adult ) cells for 48 h in the presence or absence OSMI - 1 ( 10 µM , final ) ( n = 3 biological replicates ) . The effects of Stx2a in vehicle controls were compared with culture medium prepared from HRPTEpi cells maintained in the absence of Stx2a , and OSMI - 1 treatment was compared with that of the vehicle ( DMSO ) controls in the presence of Stx2a . Data information : For graphs error bar represents mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

O O O O 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 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 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. LDH cytotoxicity assay following Stx2a (10 ng/mL) treatment of HRPTEpi (adult) cells for 48 h in the presence or absence OSMI-1 (10 µM, final) (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with culture medium prepared from HRPTEpi cells maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a. Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C . ELISAs were used to analyze the inhibitory effect of OSMI - 1 on cytokine / chemokine production from HRPTEpi cells ( adult ) exposed to Stx2a ( 10 ng / mL ) for 48 h ( n = 3 biological replicates ) . The effects of OSMI - 1 were compared with those of the vehicle ( DMSO ) control . Data information : For graphs , error bar represents mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

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

C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on cytokine/chemokine production from HRPTEpi cells (adult) exposed to Stx2a (10 ng/mL) for 48 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) control. Data information: For graphs , error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C . ELISAs were used to analyze the inhibitory effect of OSMI - 1 on IL - 8 and CCL - 2 production from 3D cultures of HRPTEpi cells exposed to Stx2a ( 10 ng / mL ) for 72 h ( n = 3 biological replicates ) . The effects of OSMI - 1 were compared with those of the vehicle ( DMSO ) controls . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

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

C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of HRPTEpi cells exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


F . ELISAs were used to analyze the inhibitory effect of OSMI - 1 on IL - 8 and CCL - 2 production from 3D cultures of human mini - kidneys exposed to Stx2a ( 10 ng / mL ) for 72 h ( n = 3 biological replicates ) . The effects of OSMI - 1 were compared with those of the vehicle ( DMSO ) controls . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

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

F. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of human mini-kidneys exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


G , H . Human apoptosis antibody array analysis of multiple proteins using pooled lysate from 3D - human mini - kidney spheroids following Stx2a ( 10 ng / mL ) treatment for 72 h in the presence or absence OSMI - 1 ( 10 µM , final ) . ( G ) Antibody spots representing signal differences are indicated in red boxes . ( H ) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure ( n = 2 biological replicates ) . Dashed line represents the reference point of the fold change . Raw values of fluorescence intensities are provided in the Source Data . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H. Human apoptosis antibody array analysis of multiple proteins using pooled lysate from 3D-human mini-kidney spheroids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (G) Antibody spots representing signal differences are indicated in red boxes. (H) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Raw values of fluorescence intensities are provided in the Source Data. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C , D . ELISAs were used to analyze the inhibitory effect of OSMI - 1 ( 10 µM , final ) on Kidney injury molecule - 1 ( KIM - 1 ) secretion ( C ) and IL - 8 and CCL - 2 production ( D ) in culture supernatants of iPSC - derived human kidney organoids exposed to Stx2a ( 10 ng / mL ) for 72 h ( n = 3 biological replicates ) . The effects of OSMI - 1 were compared with those of the vehicle ( DMSO ) controls . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

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 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 O 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, D. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on Kidney injury molecule-1 (KIM-1) secretion (C) and IL-8 and CCL-2 production (D) in culture supernatants of iPSC-derived human kidney organoids exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


E , F . Human apoptosis antibody array analysis of multiple proteins using pooled lysates from iPSC - derived human kidney organoids following Stx2a ( 10 ng / mL ) treatment for 72 h in the presence or absence OSMI - 1 ( 10 µM , final ) . ( E ) Antibody spots representing signal differences are indicated in red boxes . ( F ) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure ( n = 2 biological replicates ) . Dashed line represents the reference point of the fold change . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 .

O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F. Human apoptosis antibody array analysis of multiple proteins using pooled lysates from iPSC-derived human kidney organoids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (E) Antibody spots representing signal differences are indicated in red boxes. (F) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


B . Survival rate of mice challenged once with Stx2a ( 132 . 5 ng / kg ) and injected daily with two doses ( 300 or 1000 µg / mouse ) of OSMI - 1 ( n = 10 , 5 per dose ) or vehicle ( n = 10 ) . The effect of OSMI - 1 ( 1000 µg / mouse ) was compared with that of the vehicle control ( a log - rank test with GraphPad Prism software ) .

O O O O O O O O O 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 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. Survival rate of mice challenged once with Stx2a (132.5 ng/kg) and injected daily with two doses (300 or 1000 µg/mouse) of OSMI-1 (n = 10, 5 per dose) or vehicle (n = 10). The effect of OSMI-1 (1000 µg/mouse) was compared with that of the vehicle control (a log-rank test with GraphPad Prism software).


C . Chemical and hematological analyses of blood pooled from three mice at day 3 after Stx2a challenge . Data are presented as mean ± S . E . M . ( n = 3 biological replicates from nine mice , two - tailed Student ' s t - test ) . The effects of OSMI - 1 ( 1000 µg / mouse ) were compared with those of the vehicle control . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 .

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

C. Chemical and hematological analyses of blood pooled from three mice at day 3 after Stx2a challenge. Data are presented as mean ± S.E.M. (n = 3 biological replicates from nine mice, two-tailed Student's t-test). The effects of OSMI-1 (1000 µg/mouse) were compared with those of the vehicle control. *P < 0.05; **P < 0.01; and ***P < 0.001


( C ) Brucella replication in BMMs expressing either GFP or GFP - BspF and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria , measured as number of bacteria per cell at 24 h pi . Data are means ± SD of n = 3 independent experiments . Grey dots represent values from individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , two - way ANOVA followed by Dunnett ' s multiple comparisons test ) compared to controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Brucella replication in BMMs expressing either GFP or GFP-BspF and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments. Grey dots represent values from individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to controls.


( A ) Quantification of ss - eGFP - FKBPF36M trafficking in HeLa ( M ) - C1 cells transfected for 24 h with pmCherry ( mCherry ) or pmCherry - BspF ( mCherry - BspF ) . Rapamycin was added to initiate secretory traffic of ss - eGFP - FKBPF36M and its colocalization with calnexin ( ER ) , ERGIC - 53 ( ERGIC ) , GM130 ( Golgi ) , p230 ( TGN ) , or secretory vesicles ( SV ) was scored over a 60 min time course . Data are means ± SD from n = 3 independent experiments . Asterisks indicate statistically significant differences between mCherry - and mCherry - BspF - expressing cells as determined by a two - way ANOVA with Sidak ' s multiple comparisons test ( P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of ss-eGFP-FKBPF36M trafficking in HeLa(M)-C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry-BspF (mCherry-BspF). Rapamycin was added to initiate secretory traffic of ss-eGFP-FKBPF36M and its colocalization with calnexin (ER), ERGIC-53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) was scored over a 60 min time course. Data are means ± SD from n=3 independent experiments. Asterisks indicate statistically significant differences between mCherry- and mCherry-BspF-expressing cells as determined by a two-way ANOVA with Sidak's multiple comparisons test (P<0.05).


( F ) Representative confocal fluorescence micrographs of HeLa cells co - transfected for 24 h to produce mCherry - BspF and either GFP - Rab6a , GFP - Rab8a or Arf6 - GFP and treated with Cytochalasin D ( 200 nM ) for 30 min prior to fixation . Scale bars : 10 and 2 µm ( insets ) . ( G ) Quantification of localization of GFP - Rab6a , GFP - Rab8a and Arf6 - GFP on mCherry - BspF - labelled tubules in transfected HeLa cells . Data are means ± SD from n = 3 independent experiments , in which at least 300 individual cells per experiment were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal fluorescence micrographs of HeLa cells co-transfected for 24 h to produce mCherry-BspF and either GFP-Rab6a, GFP-Rab8a or Arf6-GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). (G) Quantification of localization of GFP-Rab6a, GFP-Rab8a and Arf6-GFP on mCherry-BspF-labelled tubules in transfected HeLa cells. Data are means ± SD from n=3 independent experiments, in which at least 300 individual cells per experiment were analyzed.


( A ) Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry - BspF ( grayscale panels ) , incubated on ice with AlexaFluor ™ 488 - Cholera Toxin subunit B ( CTxB ; green ) and shifted to 37 ˚ C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus ( stained using an anti - GM130 antibody ; purple ) . CTxB accumulation within Golgi structures appears white in overlays . Scale bars : 10 and 2 µm ( insets ) . ( B ) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry , mCherry - BspF or HA - BspF over a 30 min time course , expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker , as in ( A ) . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate statistically significant differences compared to mCherry - producing cells as determined by a two - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry-BspF (grayscale panels), incubated on ice with AlexaFluor™488-Cholera Toxin subunit B (CTxB; green) and shifted to 37˚C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti-GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). (B) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry-BspF or HA-BspF over a 30 min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared to mCherry-producing cells as determined by a two-way ANOVA with Tukey's multiple comparisons test (P<0.05). (


( D ) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA - mediated depletion of either Arf6 ( siArf6 ) , Rab8a ( siRab8a ) or Rab6a / a ' ( siRab6a / a ' ) after AlexaFluor ™ 488 - CTxB binding on ice followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . ( E ) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock - infected or infected with wild type ( 2308 ) , ΔbspF , complemented ∆ bspF ( ΔbspF : : bspF ) , ∆ bspB or complemented ∆ bspB ( ΔbspB : : bspB ) bacteria for 24 h , incubated for 30 min with AlexaFluor ™ 488 - CTxB on ice for binding followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate a statistically significant difference compared to mock - infected cells as determined by a one - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA-mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a) or Rab6a/a' (siRab6a/a') after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (E) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock-infected or infected with wild type (2308), ΔbspF, complemented ∆bspF (ΔbspF::bspF), ∆bspB or complemented ∆bspB (ΔbspB::bspB) bacteria for 24 h, incubated for 30 min with AlexaFluor™488-CTxB on ice for binding followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to mock-infected cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (


( G - I ) Brucella replication in BMMs treated with non - targeting siRNAs ( siNT ) , or siRNAs against Rab6a / a ' ( siRab6a / a ' ) ( G ) , Rab8a ( siRab8a ) ( H ) or Arf6 ( siArf6 ) ( I ) and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria , measured as number of bacteria per cell at 24 h pi . Data are means ± SD of n = 3 independent experiments in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Brucella replication in BMMs treated with non-targeting siRNAs (siNT), or siRNAs against Rab6a/a' (siRab6a/a') (G), Rab8a (siRab8a) (H) or Arf6 (siArf6) (I) and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


( B ) Representative co - immunoprecipitations of HA - BspF and myc - ACAP1 in HeLa cells . HeLa cells were transfected to either co - produce or individually produce HA - BspF and myc - ACAP1 and either HA - BspF or myc - ACAP1 were immunoprecipitated using either anti - HA - conjugated ( upper panel ) or anti - myc - conjugated ( lower panel ) magnetic beads following cross - linking ( + DSP ) or not ( - DSP ) with dithiobis [ succinimidylpropionate ] . Input lysates ( 10 % of the post - nuclear supernatant ) and co - immunoprecipitates were separated by SDS - PAGE and probed for HA - BspF and myc - ACAP1 by Western blotting .

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

(B) Representative co-immunoprecipitations of HA-BspF and myc-ACAP1 in HeLa cells. HeLa cells were transfected to either co-produce or individually produce HA-BspF and myc-ACAP1 and either HA-BspF or myc-ACAP1 were immunoprecipitated using either anti-HA-conjugated (upper panel) or anti-myc-conjugated (lower panel) magnetic beads following cross-linking (+DSP) or not (-DSP) with dithiobis[succinimidylpropionate]. Input lysates (10% of the post-nuclear supernatant) and co-immunoprecipitates were separated by SDS-PAGE and probed for HA-BspF and myc-ACAP1 by Western blotting.


( C ) Representative confocal micrographs of HeLa cells transfected to produce mCherry - BspF and GFP - ACAP1 and treated with Cytochalasin D ( 200 nM ) for 30 min and quantification of colocalization between mCherry - BspF and GFP - ACAP1 . Arrows indicate areas of BspF and ACAP1 colocalization . Scale bars : 10 µm and 2 µm ( insets ) . Data are means ± SD from n = 3 independent experiments in which 10 cells were analyzed per experiment . Pearson ' s correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc _ 2 plug - in .

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

(C) Representative confocal micrographs of HeLa cells transfected to produce mCherry-BspF and GFP-ACAP1 and treated with Cytochalasin D (200 nM) for 30 min and quantification of colocalization between mCherry-BspF and GFP-ACAP1. Arrows indicate areas of BspF and ACAP1 colocalization. Scale bars: 10 µm and 2 µm (insets). Data are means ± SD from n=3 independent experiments in which 10 cells were analyzed per experiment. Pearson's correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc_2 plug-in.


( B ) Representative Western blot analysis of co - immunoprecipitations of myc - ACAP1 and Arf6 - HA in the presence or absence of HA - BspF . HeLa cells were transfected to produce Arf6 - HA and combinations of myc - ACAP1 and HA - BspF , or not , and myc - ACAP1 was immunoprecipitated using anti - myc - conjugated magnetic beads . Input lysates ( 6 % of post - nuclear supernatants ) and co - immunoprecipitates were separated by SDS - PAGE and probed for Arf6 - HA , HA - BspF and myc - ACAP1 by Western blotting . Quantification of the Arf6 / ACAP1 ratio was performed by densitometric analysis . Data are means ± SD of 3 independent experiments . The asterisk indicates a statistically significant difference ( P = 0 . 0017 , unpaired Student ' s t test ) between BspF - producing and control conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of co-immunoprecipitations of myc-ACAP1 and Arf6-HA in the presence or absence of HA-BspF. HeLa cells were transfected to produce Arf6-HA and combinations of myc-ACAP1 and HA-BspF, or not, and myc-ACAP1 was immunoprecipitated using anti-myc-conjugated magnetic beads. Input lysates (6% of post-nuclear supernatants) and co-immunoprecipitates were separated by SDS-PAGE and probed for Arf6-HA, HA-BspF and myc-ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference (P = 0.0017, unpaired Student's t test) between BspF-producing and control conditions.


( E ) Bacterial replication in BMMs transduced to either produce GFP , GFP - ACAP1 or GFP - ACAP1R448Q and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria for 24 h . Data are means ± SD of n = 3 independent experiments , in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , two - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial replication in BMMs transduced to either produce GFP, GFP-ACAP1 or GFP-ACAP1R448Q and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


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

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


( H ) Brucella replication in BMMs treated with either non - targeting siRNAs ( siNT ) , or siRNAs against Stx6 ( siStx6 ) and infected for 24 h with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria . Data are means ± SD of n = 3 independent experiments , in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Brucella replication in BMMs treated with either non-targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


A . Immunostaining of P5 cerebellum with indicated antibodies . ( A ) low magnification . ( A ' - A ' ' ' ' ) high magnification . The solid line indicates the pial edge of the EGL . The dashed line demarcates the bottom of the KI67 - stained region . The outer EGL corresponds to the area between the solid and dashed lines , which contain GCPs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunostaining of P5 cerebellum with indicated antibodies. (A) low magnification. (A'-A'''') high magnification. The solid line indicates the pial edge of the EGL. The dashed line demarcates the bottom of the KI67-stained region. The outer EGL corresponds to the area between the solid and dashed lines, which contain GCPs.


Atoh1 KD analysis . ( G , H ) Atoh1 shRNA was introduced to P5 mice EGL together with H2B - EGFP plasmid and immunostaining for NEUROD1 ( Cyan ) and KI67 ( magenta ) was performed 24 hrs later . Nuclei of electroporated cells were labeled with GFP ( green ) . Images of Control ( G ) and Atoh1 KD ( H ) . Arrows indicate the cells of interest where GFP signals are co - localized with NEUROD1 and KI67 .

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

Atoh1 KD analysis. (G, H) Atoh1 shRNA was introduced to P5 mice EGL together with H2B-EGFP plasmid and immunostaining for NEUROD1 (Cyan) and KI67 (magenta) was performed 24 hrs later. Nuclei of electroporated cells were labeled with GFP (green). Images of Control (G) and Atoh1 KD (H). Arrows indicate the cells of interest where GFP signals are co-localized with NEUROD1 and KI67.


( I ) Proportions of NEUROD1 + , KI67 + cells ( ND + GCPs ) among the electroporated GCPs ( GFP + , KI67 + cells ) were increased in the Atoh1 KD mice . ( J ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 4 ) and Atoh1 shRNA ( sh # 1 : N = 3 , sh # 2 : N = 3 ) . Bars and error bars are representing as Mean ± SEM . * p < 0 . 05 , N . S . : Not Significant . Number of cells : 98 , 71 , 81 and 60 cells from N = 4 mice ( sclamble ) , 127 , 93 and 51 cells from N = 3 mice ( sh # 1 ) , 75 , 111 , 38 cells from N = 3 mice ( sh # 2 ) .

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

(I) Proportions of NEUROD1+, KI67+ cells (ND+GCPs) among the electroporated GCPs (GFP+, KI67+ cells) were increased in the Atoh1 KD mice. (J) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=4) and Atoh1 shRNA (sh#1: N=3, sh#2: N=3). Bars and error bars are representing as Mean ± SEM. *p < 0.05, N.S.: Not Significant. Number of cells: 98,71,81 and 60 cells from N=4 mice (sclamble), 127,93 and 51 cells from N=3 mice (sh#1), 75,111,38 cells from N=3 mice (sh#2).


B . Immunostaining of P5 cerebellum with indicated antibodies . Boxplots of the intensity of CCND1 immuno - signal ( B ' ' ' ' , normalized to the mean intensity of AT + GCPs ) . We measured the intensities of CCND1 in ATOH1 + / NEUROD1 - cells and NEUROD1 + / ATOH1 - cells . ATOH1 + / NEUROD1 + cells were not included in this quantification . These boxplots are showing the median ( central band ) , 25 % / 75 % quartiles ( boxes ) and the ranges ( lower whiskers represent the regions of lower box - 1 . 5 * IQR ( inter - quartile range ) , upper whisker represent the regions of upper box - 1 . 5 * IQR ) . Wilcoxon test were performed between AT + GCPs and ND + GCPs . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001.


C - F . Ccnd1 KD analysis . ( C , D ) Ccnd1 shRNAs together with H2B - EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 ( magenta ) and KI67 ( Cyan ) was performed the following day . Nuclei of electroporated cells were labeled with GFP ( yellow ) . Images of Control ( C ) and Ccnd1 KD ( D ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( E ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were decreased in the Ccnd1 KD mice . ( F ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 3 ) and Ccnd1 KD ( sh # 1 : N = 3 , sh # 2 : N = 3 ) mice . Bars and error bars are representing as Mean ± SEM . * * * p < 0 . 001 , N . S . : Not Significant . Number of cells : 52 , 64 and 107 cells from N = 3 mice ( scramble ) , 72 , 79 and 75 cells from N = 3 mice ( sh # 1 ) , 84 , 70 and 40 cells from N = 3 mice ( sh # 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Ccnd1 KD analysis. (C, D) Ccnd1 shRNAs together with H2B-EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (Cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (C) and Ccnd1 KD (D). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (E) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were decreased in the Ccnd1 KD mice. (F) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Bars and error bars are representing as Mean ± SEM. ***p < 0.001, N.S.: Not Significant. Number of cells: 52, 64 and 107 cells from N=3 mice (scramble), 72,79 and 75 cells from N=3 mice (sh#1), 84, 70 and 40 cells from N=3 mice (sh#2).


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

O O O O O O O O O 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 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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 assay in P7 mouse cerebellum with anti - Prox1 or goat IgG as a negative control . Primer set used in this experiment was designed to amplify Ccnd1 promoter region . M - R . Administration of Valproic acid ( VPA ) to electroporated mice . ( M ) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation . Immunostaining for CCND1 ( magenta ) was performed the day following the last VPA treatment . Nuclei of electroporated cells were detected with GFP ( green ) . Arrows indicate the cells of interest where GFP signals are co - localized with CCND1 . ( N - Q ) Images of H2B - GFP with Saline ( N ) , PROX1 with Saline ( O ) , H2B - GFP with VPA ( P ) , and PROX1 with VPA ( Q ) . ( R ) The relative proportions of CCND1 + cells among the electroporated cells ( normalized to the Control ) . Tukey - Kramer test among Control + saline ( N = 4 ) , PROX1 + saline ( N = 3 ) , Control + VPA ( N = 3 ) and PROX1 + VPA ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Number of cells : 120 , 488 , 934 and 211 from N = 4 mice ( Control + saline ) , 630 , 356 and 545 from N = 3 mice ( Control + VPA ) , 363 , 127 and 219 from N = 3 mice ( Prox1 + saline ) , 110 , 116 and 202 from N = 3 mice ( Prox1 + VPA ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the pilot group , study group 1 and study group 2 were calculated on the bases of pre - surgery MRI scans .

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


( C ) The brain was cut in 6 mm - thick slices using an adjustable brain matrix , obtaining 9 - 10 slices / brain . ( D ) Each brain slice was divided along the midline and each part subdivided in blocks . The cervical spinal cord was cut in 4 blocks .

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(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 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 .

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


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

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


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

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

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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 IHC in cortical and subcortical regions of the injected and contralateral hemispheres of NHP S2 . 3 .

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


( B ) Physiological ARSA expression in cortical and subcortical regions of the injected hemisphere in LV . GFP - injected NHP P1 . Scale bar , 100 µm . Insets show ARSA - expressing cells at higher magnification . Note the perinuclear accumulation of ARSA protein .

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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 ) Quantification of immunopositive signal in brain slices 3 to 6 ( n = 8 blocks / slice ) . Data are expressed as fold increase to mean immunopositive signal measured in matched brain regions of NHP P1 ( physiological ) . Two - way ANOVA and Bonferroni post - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs physiological .

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(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 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 ) .

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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 ) 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 .

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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 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 .

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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.