F ) Kinetics of the expression of tBID - GFP ( blue ) and PI intake upon tBID expression ( purple ) and unstransfected HCT116 AKO cells ( in grey ) . Plots show the average fluorescence intensity ( dark lines ) and its standard deviation ( shaded areas ) from 3 technical replicates and 3 independent experiments . Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.


G ) Quantification of PI + cells from total transfected cells with GFP - BAX , tBID - GFP , GFP - BCLXL and GFP . n = 3 independent experiments with > 10000 cells per condition per experiment . * * * p < 0 , 001 with respect to untransfected 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

G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition.


H ) Cell death induced by tBID - GFP in HCT AKO cells in the absence and presence of ZVAD , NSA and Ferrostatin - 1 . Data correspond to three independent experiments , n > 30 cells per condition per experiment . * * * p < 0 , 001 with respect to MOCK condition .

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 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

H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.


A ) Representative confocal immunofluorescence and bright field ( BF ) images of HCT DKO cells expressing tBID - GFP ( in green ) treated or not with COMBO ( ABT737 + S63586 ) . Cyt c shown in magenta and nuclei in cyan . Scale bar 10 µm . B ) Effect of tBID - GFP and mCherry - BCLXL on cell death , measured as percentage of cells showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 cells per condition per experiment . * * * p < 0 , 001 , * * p < 0 , 025 and * p < 0 , 05 with respect to tBID - GFP in HCT AKO 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 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 O O O O O O O O O O O O O O O O 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 immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm. B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition.


C ) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments . Nuclei ( cyan ) and cyt c ( magenta ) are shown . Scale bar 10µm . Arrowheads represent cells showing released cytochrome c and pyknotic nuclei .

O O O O O O O O O O O O O O O O O O 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 immunofluorescence images of HCT DKO cells upon indicated treatments. Nuclei (cyan) and cyt c (magenta) are shown. Scale bar 10µm. Arrowheads represent cells showing released cytochrome c and pyknotic nuclei.


E ) % of cell death induced by TRAIL ( 1µg / ml ) , COMBO and their combination at different treatment durations on HCT DKO measured as in panel B . Paired Student ' s t - test * * p < 0 , 025 and * p < 0 , 05 with respect to MOCK condition .

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

E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition.


I ) Effect of TRAIL ( 1µg / ml ) , COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells . Each dot corresponds to the average of 3 technical replicates from 4 independent experiments . * * * p < 0 , 001 .

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

I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.


J ) Representative HyVolution - superresolution images of TFAM - RFP expressing U2OS BAX / BAK DKO cells in the in the absence ( top ) and presence of tBID - GFP ( bottom ) . TFAM appears in red , TOM20 / mitochondria in grey and tBID - GFP in green .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative HyVolution - superresolution images of TFAM-RFP expressing U2OS BAX/BAK DKO cells in the in the absence (top) and presence of tBID-GFP (bottom). TFAM appears in red, TOM20/mitochondria in grey and tBID-GFP in green.


K ) Quantification of cells with TFAM release on U2OS BAX / BAK DKO cells transfected with GFP - BAX / tBID - GFP and TFAM - RFP in the presence / absence of COMBO . Dots correspond to individual experiments ( n = 3 individual experiments with more than 10 cells per condition per experiment ) . Unpaired Student ' s t - test * * * p < 0 , 001 with respect to non - treated 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

K) Quantification of cells with TFAM release on U2OS BAX/BAK DKO cells transfected with GFP-BAX/tBID-GFP and TFAM-RFP in the presence/ absence of COMBO. Dots correspond to individual experiments (n=3 individual experiments with more than 10 cells per condition per experiment). Unpaired Student's t-test *** p<0,001 with respect to non-treated conditions.


B ) Cell death induced by tBID - GFP and its variants on HCT AKO , measured as percentage of cells showing released cyt c or pyknotic nuclei . Data correspond to three independent experiments , with n > 30 cells per condition per experiment . * * * p < 0 , 001 and * p < 0 , 05 with respect to tBID - GFP WT 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

B) Cell death induced by tBID-GFP and its variants on HCT AKO, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to three independent experiments, with n> 30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to tBID-GFP WT condition.


D ) Effect of tBID wt and α6 - mutants overexpression on HCT AKO cells , measured as DRAQ7 + / GFP + cells and normalized to tBID wt condition . n = 3 independent experiments , * * p < 0 , 025 and * p < 0 , 05 with respect to tBID - GFP wt condition . E ) Effect of tBID wt and α6 - mutants overexpression on MEF BID KO cells , measured as DRAQ7 + / GFP + cells and normalized to tBID wt condition . Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification . n = 3 independent experiments * * * p < 0 , 001 with respect to tBID - GFP wt 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 O O O O O O O O O O O O O O O O O O O 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) Effect of tBID wt and α6-mutants overexpression on HCT AKO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. n=3 independent experiments, ** p<0,025 and * p<0,05 with respect to tBID-GFP wt condition. E) Effect of tBID wt and α6-mutants overexpression on MEF BID KO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification. n=3 independent experiments *** p<0,001 with respect to tBID-GFP wt condition.


A ) Representative CLEM images of mitochondria in HCT AKO cells ( control ) and HCT AKO cells expressing GFP - BAX or tBID - GFP . Black arrows indicate MOM disruptions . B - D ) Effect of GFP - BAX and tBID - GFP on HCT AKO mitochondrial structure . ( B ) Number of disruptions at the MOM per mitochondrial area , each dot represents an individual cell . ( C ) Mean area per mitochondrion at the condition tested , each dot represents an individual mitochondrion . ( D ) Analysis of cristae morphology , each dot represents an individual crista . Cristae shape calculated as length / width . Data represent n > 50 mitochondria from 3 - 5 independent cells . In B - D ) , boxes represent 96 % confidence interval , the average is represented by the line inside the box and whiskers correspond to S . D . The statistical significance was assessed by unpaired Student ' s t - test . * * * p < 0 , 001 and * * p < 0 , 025 with respect to HCT AKO or untransfected 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CLEM images of mitochondria in HCT AKO cells (control) and HCT AKO cells expressing GFP-BAX or tBID-GFP. Black arrows indicate MOM disruptions. B-D) Effect of GFP -BAX and tBID-GFP on HCT AKO mitochondrial structure. (B) Number of disruptions at the MOM per mitochondrial area, each dot represents an individual cell. (C) Mean area per mitochondrion at the condition tested, each dot represents an individual mitochondrion. (D) Analysis of cristae morphology, each dot represents an individual crista. Cristae shape calculated as length/width. Data represent n>50 mitochondria from 3-5 independent cells. In B-D), boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. The statistical significance was assessed by unpaired Student's t-test. *** p<0,001 and ** p<0,025 with respect to HCT AKO or untransfected condition.


E ) Representative confocal and STED images of GFP - BAX and tBID - GFP expressed in HCT AKO . Scale bar 1 µm for BAX images , 5 µm for tBID images . F ) Zoomed - in structures of GFP - BAX supramolecular assemblies with the shape of lines ( * double lines ) , arcs and rings in HCT AKO cells . Scale bar 100 nm .

E) Representative confocal and STED images of GFP-BAX and tBID-GFP expressed in HCT AKO. Scale bar 1 µm for BAX images, 5 µm for tBID images. F) Zoomed-in structures of GFP-BAX supramolecular assemblies with the shape of lines (*double lines), arcs and rings in HCT AKO cells. Scale bar 100 nm.


A ) representative western blot of cytosolic cyt c and SMAC in HeLa cells . B , C ) Western Blot analysis of active BAX ( B ) and BAK ( C ) , in HeLa cells treated with STS or infected with Shigella flexneri ( MOI 30 ) at different time points after infection .

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

A) representative western blot of cytosolic cyt c and SMAC in HeLa cells. B,C) Western Blot analysis of active BAX (B) and BAK (C), in HeLa cells treated with STS or infected with Shigella flexneri (MOI 30) at different time points after infection.


E ) Representative Western Blot analysis of cytosolic and total ( lysate ) cyt c and SMAC in HCT wt , HCT DKO and HCT TKO cells infected with Shigella flexneri at the indicated time points after treatment / infection .

O O O O O O O O O O O O O O O O O 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 analysis of cytosolic and total (lysate) cyt c and SMAC in HCT wt, HCT DKO and HCT TKO cells infected with Shigella flexneri at the indicated time points after treatment/infection.


A ) Caspase - 3 / 7 activity induced by ABT199 and etoposide on Nalm6 ( venetoclax - sensitive ) and Nalm6 199R ( venetoclax - resistant ) cells , normalized to untreated cells . Unpaired Student ' s t - test * * * p < 0 , 001 and * * p < 0 , 025 ( Nalm6 with respect to Nalm6 199R at different conditions ) . Dots correspond to technical replicates of n = 3 independent experiments .

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

A) Caspase-3/7 activity induced by ABT199 and etoposide on Nalm6 (venetoclax-sensitive) and Nalm6 199R (venetoclax-resistant) cells, normalized to untreated cells. Unpaired Student's t-test *** p<0,001 and ** p<0,025 (Nalm6 with respect to Nalm6 199R at different conditions). Dots correspond to technical replicates of n=3 independent experiments.


D ) Representative kinetics of cell death , represented as DRAQ7 + cells in Nalm6 199R cells ( grey ) , Nalm6 199R BID KO # 1 cells ( blue ) and Nalm6 199R BID KO # 5 cells ( purple ) upon 250 ng / ml TRAIL treatment . Plots show the average fluorescence intensity ( dark lines ) and its standard deviation ( shaded areas ) from 5 technical replicates . Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min .

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

D) Representative kinetics of cell death, represented as DRAQ7+ cells in Nalm6 199R cells (grey), Nalm6 199R BID KO #1 cells (blue) and Nalm6 199R BID KO #5 cells (purple) upon 250 ng/ml TRAIL treatment. Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 5 technical replicates. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.E


H ) Cell death induced by GFP - BAK and GFP - BAK R127H on HCT AKO , measured as DRAQ7 + / GFP + cells . Unpaired Student ' s t - test . * * * p < 0 , 001 with respect to GFP BAK wt .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell death induced by GFP-BAK and GFP-BAK R127H on HCT AKO, measured as DRAQ7+/GFP+ cells. Unpaired Student's t-test. *** p<0,001 with respect to GFP BAK wt.


I ) Effect of 24 h incubation with 250 ng / ml TRAIL on Nalm6 199R , Nalm6 199R BAK KO and Nalm6 199R BOK KO , measured as DRAQ7 + cells with respect of total cell surface and normalized to untreated conditions . Each dot corresponds to the average normalized value of 3 - 4 technical replicates in n = 3 independent experiments .

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

I) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BAK KO and Nalm6 199R BOK KO, measured as DRAQ7+ cells with respect of total cell surface and normalized to untreated conditions. Each dot corresponds to the average normalized value of 3-4 technical replicates in n=3 independent experiments.


( A ) Four fetuses ( named NW - 1 , NW - 2 , NW - 3 and NW - 4 ) with different eye morphology were retrieved at E44 by caesarean

O O O O O O O O O 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) Four fetuses (named NW-1, NW-2, NW-3 and NW-4) with different eye morphology were retrieved at E44 by caesarean section


( B ) DraI digestion of PCR products from the multiple organs of the four fetuses ( Upper ) . NC , negative control with no genomic DNA loaded . Mut , MITFL247S / L247S genomic DNA loaded . WT , LW genomic DNA loaded . Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis ( Bottom ) . Mut , MITFL247S / L247S genomic DNA loaded . WT , LW genomic DNA

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DraI digestion of PCR products from the multiple organs of the four fetuses (Upper). NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (Bottom). Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded


( A ) Representative microscopic appearances of the retina and RPE cells ( arrowhead ) of a chimeric , a WT and a Mut fetus at the same gestational age by H & E staining . WT , Bama WT fetus . Mut , Bama MITFL247S / L247S fetus . NW - 2 , the chimeric fetus . Scale bars , 100

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 microscopic appearances of the retina and RPE cells (arrowhead) of a chimeric, a WT and a Mut fetus at the same gestational age by H&E staining. WT, Bama WT fetus. Mut, Bama MITFL247S/L247S fetus. NW-2, the chimeric fetus. Scale bars, 100 μm


( B ) Representative immunofluorescence images showed the expression of multiple RPE markers Pax6 , MITF and Bestrophin in the chimeric fetuses . Blue , Hochst3342 . WT , Bama WT fetus as positive control . NW - 2 , the chimeric fetus . MITFL247S / L247S fetus as negative control . Scale bars , 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence images showed the expression of multiple RPE markers Pax6, MITF and Bestrophin in the chimeric fetuses. Blue, Hochst3342. WT, Bama WT fetus as positive control. NW-2, the chimeric fetus. MITFL247S/L247S fetus as negative control. Scale bars, 50 μm


( A ) Representative light microscopic images show that the in vitro cultured RPE cells exhibited a hexagonal shape . D3 , D5 and D11 , cultured for 3 days , 5 days and 11 days , respectively . Scale bars , 100

O O O O O O O O O O O O O O O O O O O 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 light microscopic images show that the in vitro cultured RPE cells exhibited a hexagonal shape. D3, D5 and D11, cultured for 3 days, 5 days and 11 days, respectively. Scale bars, 100 μm


Representative immunofluorescence images showed the expression of RPE - specific markers , including RPE65 ( B in the cultured RPE cells . Scale bars , 50

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

Representative immunofluorescence images showed the expression of RPE-specific markers, including RPE65 (B in the cultured RPE cells. Scale bars, 50 μm


Representative immunofluorescence images showed the expression of RPE - specific markers , includin ZO - 1 ( C ) and Bestrophin ( D ) , respectively , in the cultured RPE cells . Scale bars , 50

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

Representative immunofluorescence images showed the expression of RPE-specific markers, includin ZO-1 (C) and Bestrophin (D), respectively, in the cultured RPE cells. Scale bars, 50 μm


( E ) Semi - quantitative RT - PCR analysis of RPE functional markers in cultured porcine RPE cells . GAPDH was used to confirm the cDNA quality of all the

O O O O O O O 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) Semi-quantitative RT-PCR analysis of RPE functional markers in cultured porcine RPE cells. GAPDH was used to confirm the cDNA quality of all the samples


( F ) Western blotting identified three RPE markers in porcine RPE cells . GAPDH served as a housekeeping gene

(F) Western blotting identified three RPE markers in porcine RPE cells. GAPDH served as a housekeeping gene control


( G ) ELISA identified secreted VEGF and PEDF in chimeric and WT porcine RPE cells . Media were collected at 48 hours after the cells were seeded onto 12 - well cell culture plates to be used for analyses . The results are representative of three independent experiments and were evaluated using a two - tailed t - test . No significant differences were identified . Data are presented as the mean ± standard error mean ( SEM ) . WT , RPE cells cultured from the NW - 8 fetus . CH , RPE cells cultured from chimeric NW - 7

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA identified secreted VEGF and PEDF in chimeric and WT porcine RPE cells. Media were collected at 48 hours after the cells were seeded onto 12-well cell culture plates to be used for analyses. The results are representative of three independent experiments and were evaluated using a two-tailed t-test. No significant differences were identified. Data are presented as the mean ± standard error mean (SEM). WT, RPE cells cultured from the NW-8 fetus. CH, RPE cells cultured from chimeric NW-7 fetus


( A ) The full - term chimera ( NW - 16 ) piglet was born and showed normal development , size and morphology of the

(A) The full-term chimera (NW-16) piglet was born and showed normal development, size and morphology of the eyes


( B ) Digestion of the MITF PCR products in multiple organs of the full - term chimeric pig . NC , negative control with no genomic DNA loaded . Mut , MITFL247S / L247S genomic DNA loaded . WT , Bama GFP - labeled genomic DNA loaded . In the chimera piglet , GFP - specific primers were used to further confirm the chimeric contribution to multiple organs in the piglet . GAPDH was used to confirm the DNA quality of all the samples . NC , negative control with no genomic DNA loaded . Mut , Bama MITFL247S / L247S genomic DNA loaded . WT , Bama GFP - labeled genomic DNA

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Digestion of the MITF PCR products in multiple organs of the full-term chimeric pig. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded. In the chimera piglet, GFP-specific primers were used to further confirm the chimeric contribution to multiple organs in the piglet. GAPDH was used to confirm the DNA quality of all the samples. NC, negative control with no genomic DNA loaded. Mut, Bama MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded


( C ) Representative microscopic appearances of the corneal epithelial cells of the chimeric piglet staining by H & E . Scale bars , 100

O 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 microscopic appearances of the corneal epithelial cells of the chimeric piglet staining by H&E. Scale bars, 100 μm


( D ) Representative microscopic appearances of the retina cells of the chimeric piglet staining by H & E . Scale bars , 100

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

(D) Representative microscopic appearances of the retina cells of the chimeric piglet staining by H&E. Scale bars, 100 μm


( A ) Representative GFP - labeled blastomeres that contributed to the chimeric porcine RPE cells are shown with immunofluorescence staining . Scale bars , 25

(A) Representative GFP-labeled blastomeres that contributed to the chimeric porcine RPE cells are shown with immunofluorescence staining. Scale bars, 25 μm


( B ) Representative GFP - labeled blastomeres that contributed to the chimeric porcine corneal epithelial cells . Almost all RPE cells and corneal epithelial cells of the chimera stained with anti - GFP antibody . Scale bars , 75

O O O O O O O O O O O O O O O O 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 GFP-labeled blastomeres that contributed to the chimeric porcine corneal epithelial cells. Almost all RPE cells and corneal epithelial cells of the chimera stained with anti-GFP antibody. Scale bars, 75 μm


( C ) Representative immunofluorescence images showed the expression of an RPE marker ( BEST ) in GFP - labeled cells . Scale bars , 50

(C) Representative immunofluorescence images showed the expression of an RPE marker (BEST) in GFP-labeled cells. Scale bars, 50 μm


( D ) Representative immunofluorescence images showed the expression of a corneal epithelial cell marker ( K12 ) in GFP - labeled cells . WT , Bama WT piglet . Mut , Bama MITFL247S / L247S piglet . CH , chimeric piglet . Scale bars , 50

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

(D) Representative immunofluorescence images showed the expression of a corneal epithelial cell marker (K12) in GFP-labeled cells. WT, Bama WT piglet. Mut, Bama MITFL247S/L247S piglet. CH, chimeric piglet. Scale bars, 50 μm


A . Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine . In this and subsequent similar figures , the number of mice is indicated .

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

A. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine. In this and subsequent similar figures, the number of mice is indicated.


B . Averaged intracellular Ca2 + signals in cultured mouse DRG neurons in response to consecutive applications of 0 . 25 ppm iodine , 30 μM AITC and 2 μM capsaicin . All of the iodine responsive neurons ( n = 332 ) from 6 adult mice were included in the analysis .

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

B. Averaged intracellular Ca2+ signals in cultured mouse DRG neurons in response to consecutive applications of 0.25 ppm iodine, 30 μM AITC and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis.


C . Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine , following the intraperitoneal injection of HC030031 ( HC ) ( C ) or vehicle .

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

C. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of HC030031 (HC) (C) or vehicle.


D . Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine , following the intraperitoneal injection of AMG517 ( D ) or vehicle .

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

D. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of AMG517 (D) or vehicle.


E . Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1 - / - mice .

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

E. Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1-/-mice.


F . Quantification of the nociceptive responses in TRPA1 - / - mice within 5 min after intraplantar injection of control saline or 1 , 000 ppm iodine , following intraperitoneal injection of saline or vehicle .

O O O 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

F. Quantification of the nociceptive responses in TRPA1-/- mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of saline or vehicle.


A . Left , time course of ear swelling elicited by topical application of 5 % PVP - I solution in 0 . 15 % Oxa - challenged WT and TRPA1 - / - mice , or 5 % povidone solution in Oxa - challenged WT mice . In this and subsequent similar figures , the number of mice is indicated . Right , bar graph highlighting the response at 24 h shown at left .

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

A. Left, time course of ear swelling elicited by topical application of 5% PVP-I solution in 0.15% Oxa-challenged WT and TRPA1-/-mice, or 5% povidone solution in Oxa-challenged WT mice. In this and subsequent similar figures, the number of mice is indicated. Right, bar graph highlighting the response at 24 h shown at left.


B . Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0 . 15 % Oxa - challenged mice , and a bar graph highlighting the response at 24 h .

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

B. Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0.15% Oxa-challenged mice, and a bar graph highlighting the response at 24 h.


C . Time course of ear swelling elicited by topical application of 3 % iodine in 0 . 15 % Oxa - challenged WT and TRPA1 - / - mice , and bar graph highlighting the response at 24 h .

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

C. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged WT and TRPA1-/-mice, and bar graph highlighting the response at 24 h.


D . Hematoxylin and eosin stained tissue sections of iodine treated ear in 0 . 15 % Oxa - challenged WT and TRPA1 - / - mice ( n ≥ 3 ) .

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

D. Hematoxylin and eosin stained tissue sections of iodine treated ear in 0.15% Oxa-challenged WT and TRPA1-/-mice (n ≥ 3).


E . Time course of ear swelling elicited by topical application of 3 % iodine in 0 . 15 % Oxa - challenged TRPA1 - / - mice , following intraperitoneal injection of AMG517 or vehicle , and bar graph highlighting the response at 24 h .

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

E. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged TRPA1-/-mice, following intraperitoneal injection of AMG517 or vehicle, and bar graph highlighting the response at 24 h.


F . Time course of ear swelling elicited by topical application of 3 % iodine in 0 . 15 % Oxa - challenged mice , following intraperitoneal injection of RP67580 or vehicle , and bar graph highlighting the response at 24 h .

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

F. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of RP67580 or vehicle, and bar graph highlighting the response at 24 h.


A . Representative intracellular Ca2 + signals in hTRPA1 - expressing HEK293 cells in response to different concentrations of iodine . 2 - APB , a TRPA1 agonist , was subsequently applied to fully activate TRPA1 . RFU : relative fluorescence unit .

O O O O B-MEASURED_VAR I-MEASURED_VAR 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

A. Representative intracellular Ca2+ signals in hTRPA1-expressing HEK293 cells in response to different concentrations of iodine. 2-APB, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit.


B . Concentration - response relationships of iodine - induced intracellular Ca2 + increase in HEK293 cells expressing WT or mutant TRPA1 channels . Data are presented as mean ± s . e . m . n ≥ 8 for each construct at each concentration . The smooth curves are fits to the Hill equation .

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

B. Concentration-response relationships of iodine-induced intracellular Ca2+ increase in HEK293 cells expressing WT or mutant TRPA1 channels. Data are presented as mean ± s.e.m. n ≥ 8 for each construct at each concentration. The smooth curves are fits to the Hill equation.


C . Time course of iodine - induced currents in hTRPA1 - expressing Xenopus oocytes . HC :

C. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. HC: HC030031


D . Concentration - response relationship of iodine - induced currents in hTRPA1 - expressing Xenopus oocytes . Data are presented as mean ± s . e . m . n ≥ 8 for each concentration . The smooth curve is a fit to the Hill equation .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Concentration-response relationship of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. Data are presented as mean ± s.e.m. n ≥ 8 for each concentration. The smooth curve is a fit to the Hill equation.


E . Time course of intracellular iodine - induced macroscopic currents in an inside - out patch from hTRPA1 - expressing HEK293 cell ( n = 3 ) .

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

E. Time course of intracellular iodine-induced macroscopic currents in an inside-out patch from hTRPA1-expressing HEK293 cell (n = 3).


F . Time course of iodine - induced currents in a hTRPA1 - expressing Xenopus oocyte , which presents the current reduction upon DTT treatment ( n = 3 ) .

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

F. Time course of iodine-induced currents in a hTRPA1-expressing Xenopus oocyte, which presents the current reduction upon DTT treatment (n = 3).


G , H . Comparisons of the iodine - induced whole - cell current in HEK293 cells expressing WT hTRPA1 ( G ) or the triple mutant channel ( C421S / K710R / C856S ) ( H ) ( n ≥ 4 ) .

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

G, H. Comparisons of the iodine-induced whole-cell current in HEK293 cells expressing WT hTRPA1 (G) or the triple mutant channel (C421S/K710R/C856S) (H) (n ≥ 4).


I . Representative whole - cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin ( n = 3 ) .

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

I. Representative whole-cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3).


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


Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2 - month - old mice . ( A ) In vivo cSLO examinations of the mouse retina at 1 - and 2 - weeks post injection ( WPI ) of 2E9 total vg ( in 1 µl ) of sc - CMV - eGFP packaged with parental serotype AAV2 , state - of - art AAV2 . 7m8 , novel AAV2 . GL and AAV2 . NN capsid variants .

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

Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1- and 2-weeks post injection (WPI) of 2E9 total vg (in 1 µl) of sc-CMV-eGFP packaged with parental serotype AAV2 , state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.


( B ) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI . Acquisition settings were kept constant for all samples . Scale bar : 100 μm . ONL , outer nuclear layer ; INL , inner nuclear layer .

O O O O O O O O O O O O O O O O O O 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) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI. Acquisition settings were kept constant for all samples. Scale bar: 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer.


( C ) Confocal tile - scans of retinal cross sections immunolabelled for eGFP and Hoechst . Scale bar : 500

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

(C) Confocal tile-scans of retinal cross sections immunolabelled for eGFP and Hoechst. Scale bar: 500 μm


( D - E ) Immunolabelled cross sections of mouse retina infected with AAV2 . GL and AAV2 . NN respectively , with focus on the ONL and INL , stained for eGFP , recoverin and DAPI . Scale bar : 50 μm ONL , outer nuclear layer ; INL , inner nuclear layer .

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

(D-E) Immunolabelled cross sections of mouse retina infected with AAV2.GL and AAV2.NN respectively, with focus on the ONL and INL, stained for eGFP, recoverin and DAPI. Scale bar: 50 μm ONL, outer nuclear layer; INL, inner nuclear layer.


( F ) qRT - PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2 . 7m8 , AAV2 . GL and AAV2 . NN compared to AAV2 .

O O O O O O O O O O O O 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) qRT-PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2.7m8, AAV2.GL and AAV2.NN compared to AAV2.


Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2 - month - old mice . ( A ) In vivo cSLO examinations of the mouse retina at 1 - , 2 - and 3 - weeks post injection ( WPI ) of 8E9 total vg of ss - hRho - eGFP packaged with parental serotype AAV2 , state - of - art AAV2 . 7m8 , novel AAV2 . GL and AAV2 . NN capsid variants .

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

Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1-, 2- and 3- weeks post injection (WPI) of 8E9 total vg of ss-hRho-eGFP packaged with parental serotype AAV2, state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.


( B ) qRT - PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants .

O O O O 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) qRT-PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants.


( C ) Confocal scans of retinal cross sections , with focus on the ONL , immunolabelled for DAPI , eGFP and recoverin . The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images ( noted with asterisk * ) with merged DAPI , eGFP and recoverin signal . Acquisition settings were kept constant for all samples . Scale bar : 50 μm . ONL , outer nuclear layer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 scans of retinal cross sections, with focus on the ONL, immunolabelled for DAPI, eGFP and recoverin. The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images (noted with asterisk *) with merged DAPI, eGFP and recoverin signal. Acquisition settings were kept constant for all samples. Scale bar: 50 μm. ONL, outer nuclear layer.


Vector mediated eGFP expression after intravitreal injection in 10 - month - old dog eyes . ( A ) In vivo cSLO examinations of the dog retina at four , five and six weeks post intravitreal injection of 2E11 total vg ( in 200 µl ) of sc - CMV - eGFP packaged with wildtype AAV2 ( right eye ) , AAV2 . GL ( right eye ) and AAV2 . NN ( left eye ) capsid variants .

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

Vector mediated eGFP expression after intravitreal injection in 10-month-old dog eyes. (A) In vivo cSLO examinations of the dog retina at four, five and six weeks post intravitreal injection of 2E11 total vg (in 200 µl) of sc-CMV-eGFP packaged with wildtype AAV2 (right eye), AAV2.GL (right eye) and AAV2.NN (left eye) capsid variants.


( B - C ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) stained for GFP and DAPI at 6 WPI time point . : DAPI was used a nuclear marker . GFP = eGFP ; RPE , retinal pigmented epithelium ; OS , outer segments ; ONL , outer nuclear layer ; INL , inner nuclear layer ; GCL , ganglion cell layer .

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

(B-C) Confocal scans of immunolabelled retinal cross sections (14 μm) stained for GFP and DAPI at 6 WPI time point. : DAPI was used a nuclear marker. GFP=eGFP; RPE, retinal pigmented epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.


( D - E ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) at 6 WPI time point , focusing on the photoreceptors , using antibodies against eGFP , Rhodopsin ( Rho ) and M - opsin ( MO ) . OS , outer segments ONL , outer nuclear

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

(D-E) Confocal scans of immunolabelled retinal cross sections (14 μm) at 6 WPI time point, focusing on the photoreceptors, using antibodies against eGFP, Rhodopsin (Rho) and M-opsin (MO). OS, outer segments ONL, outer nuclear layer


Vector mediated expression after intravitreal injection in ~ 3 - year old cynomolgus macaque eyes . ( A - B ) In vivo cSLO examination images collaged to provide NHP fundus overview at 8 WPI of E12 total vg ( in 38 . 9 - 100 µl ) of sc - CMV - eGFP packaged with AAV2 . GL and AAV2 . NN capsid variants , respectively .

Vector mediated expression after intravitreal injection in ~3-year old cynomolgus macaque eyes. (A-B) In vivo cSLO examination images collaged to provide NHP fundus overview at 8 WPI of E12 total vg (in 38.9-100 µl) of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants, respectively.


( C - D ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) of the fovea centralis from AAV2 . GL and AAV2 . NN treated animals , respectively .

O O O O O O O O O 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) Confocal scans of immunolabelled retinal cross sections (14 μm) of the fovea centralis from AAV2.GL and AAV2.NN treated animals, respectively.


( E ) Percentage quantification of eGFP + photoreceptors in the ONL of foveal cross sections . AAV2 . GL technical repeats n = 4 , AAV2 . NN technical repeats n = 3 .

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

(E) Percentage quantification of eGFP+ photoreceptors in the ONL of foveal cross sections. AAV2.GL technical repeats n=4, AAV2.NN technical repeats n=3.


( F - G ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) of the periphery from AAV2 . GL ( F ) and AAV2 . NN ( G ) treated retinae . ( Η - Ι ) Higher magnification confocal scans of immunolabelled peripheral retina . ONL , outer nuclear layer ; INL , inner nuclear

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Confocal scans of immunolabelled retinal cross sections (14 μm) of the periphery from AAV2.GL (F) and AAV2.NN (G) treated retinae. (Η-Ι) Higher magnification confocal scans of immunolabelled peripheral retina. ONL, outer nuclear layer; INL, inner nuclear layer


Vector mediated eGFP expression in human retinal explants . ( B ) EVOS epifluorescence images at 2x magnification of the human retinal explants at three , six and nine days in vitro ( DIV ) after transduction with 1E11 total vg of sc - CMV - eGFP packaged with AAV2 . GL and AAV2 . NN capsid variants . White signal indicates eGFP .

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

Vector mediated eGFP expression in human retinal explants. (B) EVOS epifluorescence images at 2x magnification of the human retinal explants at three, six and nine days in vitro (DIV) after transduction with 1E11 total vg of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants. White signal indicates eGFP.


( C ) Confocal scans of day 10 tissue sections immunolabelled for eGFP and Hoechst . Scale bar : 50 μm .

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

(C) Confocal scans of day 10 tissue sections immunolabelled for eGFP and Hoechst. Scale bar: 50 μm.


( A ) Representative photopic ERG flicker series at 1 , 5 and 10 Hz with 3 cd . s / m2 illumination at 10 WPI . Arrowheads indicate light stimulus .

O O O O O O O O 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 photopic ERG flicker series at 1, 5 and 10 Hz with 3 cd.s/m2 illumination at 10 WPI. Arrowheads indicate light stimulus.


( B ) Representative photopic single flash ERG signal with 1 , 3 and 10 cd . s / m2 illumination . The vertical dotted line indicates light stimulus . ( C ) Quantification of photopic B - wave amplitude from single flash responses to 1 , 3 and 10 cd . s / m2 illumination of wildtype C57BL6 / J x 129 / sv eyes ( WT , n = 3 ) compared to treated eyes ( IVT / GL , n ≥ 3 ) and untreated eyes ( UN , n ≥ 3 ) at 10 weeks post injection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 photopic single flash ERG signal with 1, 3 and 10 cd.s/m2 illumination. The vertical dotted line indicates light stimulus. (C) Quantification of photopic B-wave amplitude from single flash responses to 1, 3 and 10 cd.s/m2 illumination of wildtype C57BL6/J x 129/sv eyes (WT, n = 3) compared to treated eyes (IVT/GL, n ≥ 3) and untreated eyes (UN, n ≥ 3) at 10 weeks post injection.


( E - F ) Confocal images of sections proximal to the injection site in Q2 ( E ) and distal to the injection site in Q4 ( F ) , showing Cnga3 expression in PNA - positive cone outer segments . OS , outer segment layer ; ONL , outer nuclear layer ; INL , inner nuclear layer .

(E-F) Confocal images of sections proximal to the injection site in Q2 (E) and distal to the injection site in Q4 (F), showing Cnga3 expression in PNA-positive cone outer segments. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.


( G - I ) Confocal images of Cnga3 - / - treated ( G ) , wildtype ( H ) and Cnga3 - / - untreated ( I ) retinal sections stained for Cnga3 . Note some non - specific signal detected in INL and adjacent synaptic layer . Scale bar : 50 μm . OS , outer segment layer ; ONL , outer nuclear layer ; INL , inner nuclear layer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of Cnga3-/- treated (G), wildtype (H) and Cnga3-/- untreated (I) retinal sections stained for Cnga3. Note some non-specific signal detected in INL and adjacent synaptic layer. Scale bar: 50 μm. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.


C - D ) Representative western blots and respective densitometries ( right panel ) for the selected SWI / SNF / BAF complex subunits ( n = 3 - 5 biological replicates ) . Arrow heads indicate non - specific bands , while # symbol highlights BAF53B .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blots and respective densitometries (right panel) for the selected SWI/SNF/BAF complex subunits (n=3-5 biological replicates). Arrow heads indicate non-specific bands, while # symbol highlights BAF53B.


F ) Representative immunoblots of co - immunoprecipitation ( co - IP ) experiments in eNPCs ( top panels ) and smNPCs ( bottom panels ) . Co - IPs were performed with antibodies against BAF170 ( left ) and BRG1 ( right ) . Inputs are homogenates from eNPCs ( left ) and smNPCs ( right ) . IgG indicates IgG - bound Sepharose beads ( as controls ) , while IP indicates immunoprecipitated samples after elution from the beads . Arrow head indicates BCL7A band at the correct MW .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoblots of co-immunoprecipitation (co-IP) experiments in eNPCs (top panels) and smNPCs (bottom panels). Co-IPs were performed with antibodies against BAF170 (left) and BRG1(right). Inputs are homogenates from eNPCs (left) and smNPCs (right). IgG indicates IgG-bound Sepharose beads (as controls), while IP indicates immunoprecipitated samples after elution from the beads. Arrow head indicates BCL7A band at the correct MW.


G ) Immunofluorescence staining of BCL7A ( purple ) along with actin , nestin and β - III tubulin ( green ) as well as GFAP ( white ) in iPSCs , smNPCs , and smNPC - derived neurons and astrocytes , respectively . Arrow heads indicate GFAP - positive glial cells with low BCL7A expression .

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

G) Immunofluorescence staining of BCL7A (purple) along with actin, nestin and β-III tubulin (green) as well as GFAP (white) in iPSCs, smNPCs, and smNPC-derived neurons and astrocytes, respectively. Arrow heads indicate GFAP-positive glial cells with low BCL7A expression.


H ) Western blot analysis of BCL7A and BAF170 in cortex and hippocampus of embryonic ( E17 . 5 ) , early postnatal ( PD0 and PD7 ) and adult ( PD120 ) mouse brain tissue showing a progressive temporal reduction of protein levels in the analyzed tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of BCL7A and BAF170 in cortex and hippocampus of embryonic (E17.5), early postnatal (PD0 and PD7) and adult (PD120) mouse brain tissue showing a progressive temporal reduction of protein levels in the analyzed tissues.


I ) Immunofluorescence staining of BCL7A ( purple ) , EdU ( green ) and NeuN ( white ) in embryonic ( E17 . 5 ) and adult ( PD120 ) hippocampal brain sections . EdU was used to label proliferating NPCs , whereas NeuN was used as a neuronal marker .

I) Immunofluorescence staining of BCL7A (purple), EdU (green) and NeuN (white) in embryonic (E17.5) and adult (PD120) hippocampal brain sections. EdU was used to label proliferating NPCs, whereas NeuN was used as a neuronal marker.


A ) Immunoblot and immunofluorescence staining of BCL7A ( red ) in eNPCs isolated from Bcl7awt / wt ( wt ) and Bcl7ako / ko ( ko ) embryos ( E13 . 5 ) . Nestin ( green ) was used as NPC marker , whereas DAPI ( blue ) was used to stain nuclei .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot and immunofluorescence staining of BCL7A (red) in eNPCs isolated from Bcl7awt/wt (wt) and Bcl7ako/ko (ko) embryos (E13.5). Nestin (green) was used as NPC marker, whereas DAPI (blue) was used to stain nuclei.


B ) Immunoblot and immunofluorescence staining of BCL7A in smNPCs . Two different BCL7A knockout clones ( KO1 and KO2 ) were used along with the parental ( P ) cell line .

B) Immunoblot and immunofluorescence staining of BCL7A in smNPCs. Two different BCL7A knockout clones (KO1 and KO2) were used along with the parental (P) cell line.


C - D ) Representative western blots and respective densitometries ( right panel ) for selected SWI / SNF / BAF complex subunits in ( C ) eNPCs ( n = 4 biological replicates ) and ( D ) smNPCs ( n = 3 - 6 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blots and respective densitometries (right panel) for selected SWI/SNF/BAF complex subunits in (C) eNPCs (n=4 biological replicates) and (D) smNPCs (n=3-6 biological replicates).


E - G ) BCL7A KO and wt smNPCs were used for ( F ) nuclear isolation followed by density sedimentation Top panels show representative immunoblots for selected SWI / SNF / BAF complex subunits and respective densitometries are shown below ( n = 3 biological replicates ) .

E-G) BCL7A KO and wt smNPCs were used for (F) nuclear isolation followed by density sedimentation Top panels show representative immunoblots for selected SWI/SNF/BAF complex subunits and respective densitometries are shown below (n=3 biological replicates).


BCL7A KO and wt smNPCs were used for differential salt extraction . Top panels show representative immunoblots for selected SWI / SNF / BAF complex subunits and respective densitometries are shown below ( n = 3 biological replicates ) .

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

BCL7A KO and wt smNPCs were used for differential salt extraction. Top panels show representative immunoblots for selected SWI/SNF/BAF complex subunits and respective densitometries are shown below (n=3 biological replicates).


H - I ) Heatmaps of SMARCA4 / BRG1 and H3K27me3 ChIP - seq in wt and BCL7A KO eNPCs . J ) Average density plots for SMARCA4 / BRG1 occupancy around the TSS in wt and BCL7A KO eNPCs .

O O O O O O O O O O O O O O O O O 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-I) Heatmaps of SMARCA4/BRG1 and H3K27me3 ChIP-seq in wt and BCL7A KO eNPCs. J) Average density plots for SMARCA4/BRG1 occupancy around the TSS in wt and BCL7A KO eNPCs.


K - L ) Heatmaps of SMARCA4 / BRG1 and H3K27me3 ChIP - seq in parental and BCL7A KO smNPCs . M ) Average density plots for SMARCA4 / BRG1 occupancy around the TSS in parental and BCL7A KO smNPCs .

K-L) Heatmaps of SMARCA4/BRG1 and H3K27me3 ChIP-seq in parental and BCL7A KO smNPCs. M) Average density plots for SMARCA4/BRG1 occupancy around the TSS in parental and BCL7A KO smNPCs.


A ) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs . Samples were collected at the indicated differentiation time points . Respective densitometries are shown on the right ( n = 6 - 7 biological replicates ) .

O O O O O O O O O O O O O O O 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) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs. Samples were collected at the indicated differentiation time points. Respective densitometries are shown on the right (n=6-7 biological replicates).


B ) Sholl analysis of β - III tubulin - positive immature wt and BCL7A KO neurons following 7 days of spontaneous eNPC differentiation ( n = 4 biological replicates , each experiment with 10 - 15 cells per 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

B) Sholl analysis of β-III tubulin-positive immature wt and BCL7A KO neurons following 7 days of spontaneous eNPC differentiation (n=4 biological replicates, each experiment with 10-15 cells per condition).


C ) Immunofluorescence staining ( central panel ) and quantification ( right panel ) of β - III tubulin - and GFAP - positive cells as well as RT - PCR analysis ( right panels ) of spontaneously differentiated iPSC - derived parental and BCL7A KO smNPCs ( n = 3 - 7 biological replicates ) .

C) Immunofluorescence staining (central panel) and quantification (right panel) of β-III tubulin- and GFAP-positive cells as well as RT-PCR analysis (right panels) of spontaneously differentiated iPSC-derived parental and BCL7A KO smNPCs (n=3-7 biological replicates).


D ) Immunofluorescence staining of BCL7A expression in hippocampal sections from adult control and Bcl7afl / fl ; Nestin - Cretg / wt mice .

O 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) Immunofluorescence staining of BCL7A expression in hippocampal sections from adult control and Bcl7afl/fl; Nestin-Cretg/wt mice.


E ) EdU experiments in adult mice . Animals were injected with EdU twice daily for three days and sacrificed 21 days thereafter . Images show immunofluorescence stainings for EdU ( green ) , doublecortin ( DCX , white ) , and S100β ( red ) in hippocampal brain sections from Bcl7awt / wt ; Nestin - Cretgt / wt ( as control ) and Bcl7afl / fl ; Nestin - Cretg / wt mice . Representative EdU + DCX + ( yellow triangles ) or EdU + S100β + ( yellow asterisk ) double - labelled cells are indicated in the insets . Scale bar = 50 μm and 20 μm ( for insets ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EdU experiments in adult mice. Animals were injected with EdU twice daily for three days and sacrificed 21 days thereafter. Images show immunofluorescence stainings for EdU (green), doublecortin (DCX, white), and S100β (red) in hippocampal brain sections from Bcl7awt/wt; Nestin-Cretgt/wt (as control) and Bcl7afl/fl; Nestin-Cretg/wt mice. Representative EdU+ DCX+ (yellow triangles) or EdU+ S100β+ (yellow asterisk) double-labelled cells are indicated in the insets. Scale bar= 50 μm and 20 μm (for insets).


F ) Quantification of EdU + ( right panel ) , EdU + DCX + ( middle panel ) and EdU + S100β + ( left panel ) cells within the hippocampal dentate gyrus ( DG ) of adult control ( n = 4 ) and Bcl7afl / fl ; Nestin - Cretg / wt ( n = 5 ) animals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of EdU+ (right panel), EdU+ DCX+ (middle panel) and EdU+ S100β+ (left panel) cells within the hippocampal dentate gyrus (DG) of adult control (n=4) and Bcl7afl/fl; Nestin-Cretg/wt (n=5) animals.


G ) Immunofluorescence staining for DCX + cells within the DG region of adult Bcl7afl / fl ; Nestin - Crewt / wt ( as control ) and Bcl7afl / fl ; Nestin - Cretg / wt animals . Confocal imaging analysis shows a lower number , misalignment and reduced neuritic complexity of DCX + labelled cells in BCL7A KO mice compared to controls . Yellow arrows indicate misaligned DCX + cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence staining for DCX+ cells within the DG region of adult Bcl7afl/fl; Nestin-Crewt/wt (as control) and Bcl7afl/fl; Nestin-Cretg/wt animals. Confocal imaging analysis shows a lower number, misalignment and reduced neuritic complexity of DCX+ labelled cells in BCL7A KO mice compared to controls. Yellow arrows indicate misaligned DCX+ cells.


H ) Immunofluorescence staining of BCL7A ( red ) of hippocampal sections from Bcl7afl / fl ; Baf53b - Crewt / wt ( as control ) and Bcl7afl / fl ; Baf53b - Cretg / wt mice .

O O O O O O O O O O O O O O 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) Immunofluorescence staining of BCL7A (red) of hippocampal sections from Bcl7afl/fl; Baf53b-Crewt/wt (as control) and Bcl7afl/fl; Baf53b-Cretg/wt mice.


I ) Immunofluorescence staining of BCL7A ( red ) and DCX ( green ) in hippocampal sections of control and Bcl7afl / fl ; Baf53b - Cretg / wt mice . Yellow arrow indicates dividing cells , whereas yellow asterisks mark differentiating immature neurons .

O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence staining of BCL7A (red) and DCX (green) in hippocampal sections of control and Bcl7afl/fl; Baf53b-Cretg/wt mice. Yellow arrow indicates dividing cells, whereas yellow asterisks mark differentiating immature neurons.


J ) Immunofluorescence staining for DCX ( white ) within the DG region of adult control and Bcl7afl / fl ; Baf53b - Cretg / wt mice . Representative images show no obvious difference in number , morphology or alignment of DCX + labelled cells . Quantification of DCX + labelled cells in control and Bcl7afl / fl ; Baf53b - Cretg / wt mice is shown on the right ( n = 3 per genotype ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence staining for DCX (white) within the DG region of adult control and Bcl7afl/fl; Baf53b-Cretg/wt mice. Representative images show no obvious difference in number, morphology or alignment of DCX+ labelled cells. Quantification of DCX+ labelled cells in control and Bcl7afl/fl; Baf53b-Cretg/wt mice is shown on the right (n=3 per genotype).


B - C ) Representative Ca2 + measurements in ( B ) wt vs BCL7A KO cortical neurons ( CN , 10 days in vitro ) and in ( C ) smNPC - derived parental and BCL7A KO neurons . Ca2 + changes were detected using Fluo - 4 and upon treatment with ( B ) 50 μM glutamate ( final concentration ) and ( C ) 100 μM glutamate ( final concentration ) . Upper right panels report Ca2 + peaks ( F ) relative to baseline ( F0 ) . In B , wt : n = 18 cells ; ko : n = 23 cells ( n = 2 independent experiments ) . In C , P : n = 27 cells ; KO1 : n = 20 cells ; KO2 : n = 19 cells ( n = 2 independent experiments ) .

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

B-C) Representative Ca2+ measurements in (B) wt vs BCL7A KO cortical neurons (CN, 10 days in vitro) and in (C) smNPC-derived parental and BCL7A KO neurons. Ca2+ changes were detected using Fluo-4 and upon treatment with (B) 50 μM glutamate (final concentration) and (C) 100 μM glutamate (final concentration). Upper right panels report Ca2+ peaks (F) relative to baseline (F0). In B, wt: n=18 cells; ko: n=23 cells (n=2 independent experiments). In C, P: n=27 cells; KO1: n=20 cells; KO2: n=19 cells (n=2 independent experiments).


D ) Immunoblot analysis of hippocampal tissues . Densitometries are shown on the right ( ctr : n = 4 - 5 mice ; ko : n = 3 - 4 mice ) .

O O O O O O O O O O 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) Immunoblot analysis of hippocampal tissues. Densitometries are shown on the right (ctr: n=4-5 mice; ko: n=3-4 mice).


E ) RT - PCR analysis of FOS expression upon glutamate ( 100 μM for 10 min ) stimulation in smNPC - derived parental and BCL7A KO neurons ( n = 5 - 6 biological replicates ) .

E) RT-PCR analysis of FOS expression upon glutamate (100 μM for 10 min) stimulation in smNPC-derived parental and BCL7A KO neurons (n=5-6 biological replicates).


F ) Immunofluorescence staining of cFos in the dentate gyrus of adult Bcl7afl / fl ; Baf53b - Cretg / wt ( ko ) mice and control ( ctr ) littermates . Mice were either kept in standard housing conditions ( upper images ; ctr : n = 3 mice ; ko : n = 3 mice ) or exposed to an enriched environment ( lower images ; ctr : n = 3 mice ; ko : n = 4 mice ) . Quantification of cFos + cells are shown below .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence staining of cFos in the dentate gyrus of adult Bcl7afl/fl; Baf53b-Cretg/wt (ko) mice and control (ctr) littermates. Mice were either kept in standard housing conditions (upper images; ctr: n=3 mice; ko: n=3 mice) or exposed to an enriched environment (lower images; ctr: n=3 mice; ko: n=4 mice). Quantification of cFos+ cells are shown below.


G - K ) Behavioral characterization of Bcl7afl / fl ; Nestin - Cretg / wt ( ko ) mice compared to control ( ctr ) littermates . Animals were tested for their locomotor activity and coordination in ( H ) the open field ( ctr : n = 13 mice ; ko : n = 11 mice ) and ( I ) RotaRod test ( ctr : n = 12 mice ; ko : n = 7 mice ) . Cognitive function was assessed in ( J ) the Y - maze spontaneous alternation task ( ctr : n = 13 mice ; ko : n = 11 mice ) and ( K ) fear conditioning paradigm ( ctr : n = 11 mice ; ko : n = 10 mice ) . The percentage of spontaneous alternations and freezing were used as measures for working and long - term memory function , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Behavioral characterization of Bcl7afl/fl; Nestin-Cretg/wt (ko) mice compared to control (ctr) littermates. Animals were tested for their locomotor activity and coordination in (H) the open field (ctr: n=13 mice; ko: n=11 mice) and (I) RotaRod test (ctr: n=12 mice; ko: n=7 mice). Cognitive function was assessed in (J) the Y-maze spontaneous alternation task (ctr: n=13 mice; ko: n=11 mice) and (K) fear conditioning paradigm (ctr: n=11 mice; ko: n=10 mice). The percentage of spontaneous alternations and freezing were used as measures for working and long-term memory function, respectively.


L - Q ) Behavioral characterization of adult Bcl7afl / fl ; Baf53b - Cretg / wt ( nKO ) mice compared to control ( nKO ) littermates . Locomotor activity and coordination were assessed in ( M ) the open field ( ctr : n = 14 mice ; nKO : n = 12 mice ) and ( N ) RotaRod paradigm ( ctr : n = 16 mice ; nKO : n = 12 mice ) . Cognitive function was tested in ( O ) the Y - maze ( ctr : n = 11 mice ; nKO : n = 11 mice ) and ( P - Q ) Barnes maze test ( ctr : n = 11 mice ; nKO : n = 11 mice ) . The percentage of spontaneous alternations and freezing were used as measures for working and long - term memory function , respectively . Representative heatmaps of Barnes maze test are reported on the right . The time spent in target ( T ) and non - target quadrants during the probe trial are shown on the right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-Q) Behavioral characterization of adult Bcl7afl/fl; Baf53b-Cretg/wt (nKO) mice compared to control (nKO) littermates. Locomotor activity and coordination were assessed in (M) the open field (ctr: n=14 mice; nKO: n=12 mice) and (N) RotaRod paradigm (ctr: n=16 mice; nKO: n=12 mice). Cognitive function was tested in (O) the Y-maze (ctr: n=11 mice; nKO: n=11 mice) and (P-Q) Barnes maze test (ctr: n=11 mice; nKO: n=11 mice). The percentage of spontaneous alternations and freezing were used as measures for working and long-term memory function, respectively. Representative heatmaps of Barnes maze test are reported on the right. The time spent in target (T) and non-target quadrants during the probe trial are shown on the right.


A ) Heatmap of significantly dysregulated genes in BCL7A KO compared to wt eNPCs .

A) Heatmap of significantly dysregulated genes in BCL7A KO compared to wt eNPCs.


E ) Heatmap of significantly dysregulated genes encoding components of the Wnt and Notch signaling pathways .

E) Heatmap of significantly dysregulated genes encoding components of the Wnt and Notch signaling pathways.


F ) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs in presence of 1 μM DAPT or 3 μM CHIR99021 .

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

F) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs in presence of 1 μM DAPT or 3 μM CHIR99021.


G ) Immunofluorescence staining of eNPCs undergoing spontaneous differentiation for 7 days . BCL7A KO eNPCs were incubated for 72 h ( 24 h in proliferation plus 48 h in differentiation medium ) with DMSO , 1 μM DAPT , 3 μM CHIR99021 or a combination of 1 μM DAPT + 3 μM CHIR99021 . Quantification of the β - III tubulin - positive or GFAP - positive cells is shown on the right ( n = 3 - 6 technical replicates from 3 independent experiments ) .

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

G) Immunofluorescence staining of eNPCs undergoing spontaneous differentiation for 7 days. BCL7A KO eNPCs were incubated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with DMSO, 1 μM DAPT, 3 μM CHIR99021 or a combination of 1 μM DAPT + 3 μM CHIR99021. Quantification of the β-III tubulin-positive or GFAP-positive cells is shown on the right (n=3-6 technical replicates from 3 independent experiments).


H ) Sholl analysis of β - III tubulin - positive immature neurons after 7 days of spontaneous differentiation ( n = 4 biological replicates with > 30 cells per 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

H) Sholl analysis of β-III tubulin-positive immature neurons after 7 days of spontaneous differentiation (n=4 biological replicates with >30 cells per condition).


I ) mRNA expression levels of TUBB3 and GFAP at 15 and 25 days of spontaneously differentiating BCL7A KO compared parental smNPCs . BCL7A KO smNPCs were treated with DAPT , CHIR99021 , DAPT + CHIR99021 or DMSO ( as control = ctr ) during the initial 7 days of spontaneous differentiation ( n = 3 - 7 biological replicates ) .

O O O O O 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 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

I) mRNA expression levels of TUBB3 and GFAP at 15 and 25 days of spontaneously differentiating BCL7A KO compared parental smNPCs. BCL7A KO smNPCs were treated with DAPT, CHIR99021, DAPT + CHIR99021 or DMSO (as control= ctr) during the initial 7 days of spontaneous differentiation (n=3-7 biological replicates).


J ) Immunoblots for non - phospho ( active ) β - catenin and TCF4 / TCF7L2 in proliferating and 48 h - differentiated wt and BCL7A KO eNPCs . Respective densitometries are shown on the right ( n = 5 - 8 biological replicates per 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

J) Immunoblots for non-phospho (active) β-catenin and TCF4/TCF7L2 in proliferating and 48 h-differentiated wt and BCL7A KO eNPCs. Respective densitometries are shown on the right (n=5-8 biological replicates per condition).