Figure 3 ( A - B ) Western blot analysis with cell lysate of uninfected and S . flexneri infected ( MOI 1 : 25 , 8 h ) ( A ) control and ATG5 siRNA knockdown THP - 1 cells , ( B ) wild type ( WT ) and ATG5 knockout ( ATG5 - / - ) MEF cells with indicated antibodies . Densitometric analysis was performed using Image J software . FC , fold change . ( C ) Left panel , representative high - content microscopy images of RIPosomes in HEK293T cells that are control cells or cells treated with rapamycin ( 500 nM , 4h ) or cells treated with rapamycin ( 500 nM , 4h ) and bafilomycin A1 ( BafA1 , 300 nM ) . Right panel , the graph depicts the average number of RIPosomes / cell . About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * p < 0 . 005 and , * * * p < 0 . 0005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) . ( D ) Left panel , representative high - content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP - 1 cells infected with S . flexneri ( 8 h ) . Right panel , the graph depicts average number of RIPosomes / cell . About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * * p < 0 . 0005 , Student ' s unpaired t - test . ( E ) The soluble and insoluble fractions of S . flexneri infected ( MOI 1 : 25 , 8 h ) control and ATG5 siRNA knockdown THP - 1 cells were subjected to Western blot analysis with indicated antibodies . ( F ) Representative confocal images of HEK293T cells transfected with GFP - RIPK2 and immunostained with anti - FK2 antibodies . Line profile : co - localization analysis using line intensity profiles . Scale bar , 3 µm . ( G ) Autophagy receptor screen using co - immunoprecipitation ( Co - IP ) assay to analyze the interaction between GFP - RIPK2 and Flag - p62 or Flag - NBRI or Flag - NDP52 or Flag - TAX1BP1 or Flag - OPTINEURIN in HEK293T cell lysate . ( H ) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S . flexneri infected THP - 1 cells ( MOI 1 : 25 , 8 h ) . ( I ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 ( 6 h ) immunostained with p62 and LC3B antibodies . Zoom panels are digital magnifications . ( J ) GST pull - down assay using purified GST or GST - RIPK2 proteins with in - vitro translated 35S radiolabeled myc - ULK1 or myc - ATG16L or myc - BECLIN1 . Representative confocal images of HEK293T cells transfected with GFP - RIPK2 ( 9 h ) and immunofluorescence assay performed with antibodies specific to ( K ) ULK1 Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications . Representative confocal images of HEK293T cells transfected with GFP - RIPK2 or mcherry - RIPK2 ( 9 h ) and immunofluorescence assay performed with antibodies specific to ( L ) ATG16L ( M ) BECLIN1 . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications . ( N ) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF - κB luciferase reporter vector pGL4 . 32 NFκB - RE , GFP - RIPK2 , and Flag - NOD1 plasmids . Mean ± SD , n = 3 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) .

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

Figure 3(A-B) Western blot analysis with cell lysate of uninfected and S. flexneri infected (MOI 1:25, 8 h) (A) control and ATG5 siRNA knockdown THP-1 cells, (B) wild type (WT) and ATG5 knockout (ATG5-/-) MEF cells with indicated antibodies. Densitometric analysis was performed using Image J software. FC, fold change.(C) Left panel, representative high-content microscopy images of RIPosomes in HEK293T cells that are control cells or cells treated with rapamycin (500 nM, 4h) or cells treated with rapamycin (500 nM, 4h) and bafilomycin A1 (BafA1, 300 nM). Right panel, the graph depicts the average number of RIPosomes/cell. About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), **p < 0.005 and, ***p < 0.0005, ordinary one-way ANOVA (Tukey's multiple comparisons test).(D) Left panel, representative high-content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP-1 cells infected with S. flexneri (8 h). Right panel, the graph depicts average number of RIPosomes/cell. About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ***p < 0.0005, Student's unpaired t-test.(E) The soluble and insoluble fractions of S. flexneri infected (MOI 1:25, 8 h) control and ATG5 siRNA knockdown THP-1 cells were subjected to Western blot analysis with indicated antibodies.(F) Representative confocal images of HEK293T cells transfected with GFP-RIPK2 and immunostained with anti-FK2 antibodies. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm.(G) Autophagy receptor screen using co-immunoprecipitation (Co-IP) assay to analyze the interaction between GFP-RIPK2 and Flag-p62 or Flag-NBRI or Flag-NDP52 or Flag-TAX1BP1 or Flag-OPTINEURIN in HEK293T cell lysate.(H) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S. flexneri infected THP-1 cells (MOI 1:25, 8 h).(I) Representative confocal images of HEK293T cells expressing GFP-RIPK2 (6 h) immunostained with p62 and LC3B antibodies. Zoom panels are digital magnifications.(J) GST pull-down assay using purified GST or GST-RIPK2 proteins with in-vitro translated 35S radiolabeled myc-ULK1 or myc-ATG16L or myc-BECLIN1.Representative confocal images of HEK293T cells transfected with GFP-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (K) ULK1 Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.Representative confocal images of HEK293T cells transfected with GFP-RIPK2 or mcherry-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (L) ATG16L (M) BECLIN1. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.(N) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF-κB luciferase reporter vector pGL4.32 NFκB-RE, GFP-RIPK2, and Flag-NOD1 plasmids. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


Figure 4 ( A - B ) The THP - 1 cell lysates were subjected to immunoprecipitation analysis ( A ) untreated and treated with iE - DAP ( 40 μg / ml , 6 h ) , ( B ) untreated and treated with MDP ( 40 μg / ml , 6 h ) . IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies . IgG ( H ) ; IgG heavy chain . ( C - D ) Co - IP analysis of the interactions between ( C ) GFP - RIPK2 and Flag - IRGM or ( D ) Flag - NOD1 and GFP - IRGM in HEK293T cell lysates . ( E ) GST pull - down assay using purified GST and GST - IRGM and in - vitro translated 35S radiolabeled myc - NOD2 , myc - NOD1 , and myc - RIPK2 . ( F ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 and mCherry or mCherry - IRGM . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Representative confocal images of THP - 1 cells infected with RFP expressing S . flexneri , ( MOI 1 : 25 , 8 h ) and immunostained with , ( H ) IRGM and RIPK2 antibody . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . In image H , for better contrast , RFP expressing Shigella is pseudocolored to blue . ( J ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 , Flag - NOD1 , and mCherry - IRGM . Line profile : co - localization analysis using line intensity profiles . Scale bar , 3 µm . Zoom panels are digital magnifications . ( M ) A co - IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain / s of RIPK2 interacting with IRGM . Asterisk indicate the main band of overexpressed protein . ( O ) A co - IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain / s of NOD1 interacting with IRGM . Asterisk indicate the main band of overexpressed

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

Figure 4(A-B) The THP-1 cell lysates were subjected to immunoprecipitation analysis (A) untreated and treated with iE-DAP (40 μg/ml, 6 h), (B) untreated and treated with MDP (40 μg/ml, 6 h). IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies. IgG (H); IgG heavy chain.(C-D) Co-IP analysis of the interactions between (C) GFP-RIPK2 and Flag-IRGM or (D) Flag-NOD1 and GFP-IRGM in HEK293T cell lysates.(E) GST pull-down assay using purified GST and GST-IRGM and in-vitro translated 35S radiolabeled myc-NOD2, myc-NOD1, and myc-RIPK2.(F) Representative confocal images of HEK293T cells expressing GFP-RIPK2 and mCherry or mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.Representative confocal images of THP-1 cells infected with RFP expressing S. flexneri, (MOI 1:25, 8 h) and immunostained with, (H) IRGM and RIPK2 antibody. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. In image H, for better contrast, RFP expressing Shigella is pseudocolored to blue.(J) Representative confocal images of HEK293T cells expressing GFP-RIPK2, Flag-NOD1, and mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm. Zoom panels are digital magnifications.(M) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain/s of RIPK2 interacting with IRGM. Asterisk indicate the main band of overexpressed protein.(O) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain/s of NOD1 interacting with IRGM. Asterisk indicate the main band of overexpressed protein


Figure 5 ( A ) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR - Cas9 mediated IRGM partial knockout ( IRGM + / - ) HT - 29 cells ( 2 clones were tested ) . Densitometric analysis was performed using Image J software . FC , fold change . ( B ) Left panel , soluble and insoluble fractions of S . flexneri ( MOI 1 : 25 , 8 h ) infected mouse BMDMs from Irgm1 + / + and Irgm1 - / - mice were subjected to immunoblot analysis with antibodies as indicated . Right panel , Western blot analysis with the colon lysates from Irgm1 + / + and Irgm1 - / - mice with indicated antibodies . The graph indicate ratio of band intensity ( measured using ImageJ ) and actin ( n = 5 , Mean ± SD , * p < 0 . 05 , * * p < 0 . 005 , and * * * * p < 0 . 00005 , Student ' s unpaired t - test ) ( C ) Doxycycline - inducible stable GFP - RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S . flexneri ( MOI 1 : 25 , 4h ) . The cells were fixed and subjected to high - content microscopy to quantitate the number of RIPosomes formed . The graph depicts an average number of RIPosome / cell . About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test . Western blot analysis with the cell lysate of ( D ) HT - 29 clones stably expressing Flag - vector control or Flag - IRGM Densitometric analysis was performed using Image J software . FC , fold change . Western blot analysis with the cell lysate of ( E ) THP - 1 cells transiently transfected with GFP or GFP - IRGM for 6 h . Densitometric analysis was performed using Image J software . FC , fold change . ( F ) Left panel , representative high - content microscopy images of NODo - RIPosomes in HEK293T cells transfected with GFP - RIPK2 and myc - NOD2 ( upper panel ) or GFP - RIPK2 , myc - NOD2 , and Flag - IRGM ( lower panel ) . Right panel , the graph depicts average number of NODo - RIPosome / cell . About 15000 cells plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 5 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test . ( G ) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies . ( H ) Representative confocal images of HEK293T cells transfected with mCherry and GFP - RIPK2CARD or mCherry - IRGM and GFP - RIPK2CARD for 9 h . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . ( I - K ) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin , Flag , Myc , and , GFP antibodies as indicated . Densitometric analysis was performed using Image J software . FC , fold change . ( L ) Left panels , representative high - content microscopy images of HEK293T cells transfected with GFP - RIPK2 or GFP - RIPK2 and Flag - IRGM or GFP - RIPK2 and Flag - IRGM ( S47N ) . The graph depicts the average number of RIPosome / cell . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) .

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

Figure 5(A) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR-Cas9 mediated IRGM partial knockout (IRGM+/-) HT-29 cells (2 clones were tested). Densitometric analysis was performed using Image J software. FC, fold change.(B) Left panel, soluble and insoluble fractions of S. flexneri (MOI 1:25, 8 h) infected mouse BMDMs from Irgm1+/+ and Irgm1-/- mice were subjected to immunoblot analysis with antibodies as indicated. Right panel, Western blot analysis with the colon lysates from Irgm1+/+ and Irgm1-/- mice with indicated antibodies. The graph indicate ratio of band intensity (measured using ImageJ) and actin (n=5, Mean ± SD, *p < 0.05, **p < 0.005, and ****p < 0.00005, Student's unpaired t-test)(C) Doxycycline-inducible stable GFP-RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S. flexneri (MOI 1:25, 4h). The cells were fixed and subjected to high-content microscopy to quantitate the number of RIPosomes formed. The graph depicts an average number of RIPosome/cell. About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test.Western blot analysis with the cell lysate of (D) HT-29 clones stably expressing Flag-vector control or Flag-IRGM Densitometric analysis was performed using Image J software. FC, fold change.Western blot analysis with the cell lysate of (E) THP-1 cells transiently transfected with GFP or GFP-IRGM for 6 h. Densitometric analysis was performed using Image J software. FC, fold change.(F) Left panel, representative high-content microscopy images of NODo-RIPosomes in HEK293T cells transfected with GFP-RIPK2 and myc-NOD2 (upper panel) or GFP-RIPK2, myc-NOD2, and Flag-IRGM (lower panel). Right panel, the graph depicts average number of NODo-RIPosome/cell. About 15000 cells plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=5 (biological replicates), ****p < 0.00005, Student's unpaired t-test.(G) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies.(H) Representative confocal images of HEK293T cells transfected with mCherry and GFP-RIPK2CARD or mCherry-IRGM and GFP-RIPK2CARD for 9 h. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.(I-K) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin, Flag, Myc, and, GFP antibodies as indicated. Densitometric analysis was performed using Image J software. FC, fold change.(L) Left panels, representative high-content microscopy images of HEK293T cells transfected with GFP-RIPK2 or GFP-RIPK2 and Flag-IRGM or GFP-RIPK2 and Flag-IRGM (S47N). The graph depicts the average number of RIPosome/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


Figure 7 ( C ) Left panel , representative pictures of colons of Irgm1 + / + and Irgm - / - mice untreated or treated with GSK583 infected with S . flexneri . Right panel , the graph depicts the average colon lengths of the mice groups . Mean ± SD , n = 6 , * * p < 0 . 005 , * * * p < 0 . 0005 , Student ' s unpaired t - test . ( D ) Representative microscopic images of H & E staining of colon tissues of Irgm1 + / + and Irgm - / - mice untreated or treated with GSK583 administrated with DSS . The graph depicts the combined histological scores . Mean ± SD , n = 3 ( DSS ) , * * p < 0 . 005 , Student ' s unpaired t - test . Scale bar , 200 µm ( E ) The soluble and insoluble fractionations of lysates from colon tissues of Shigella - infected or DSS - treated Irgm1 + / + and Irgm - / - mice treated with GSK583 as indicated , were subjected to immunoblot analysis with indicated antibodies . ( H - I ) Western blot analysis with the colon tissue lysates of DSS - treated or S . flexneri infected mice groups as indicated .

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

Figure 7(C) Left panel, representative pictures of colons of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 infected with S. flexneri. Right panel, the graph depicts the average colon lengths of the mice groups. Mean ± SD, n=6, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.(D) Representative microscopic images of H&E staining of colon tissues of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 administrated with DSS. The graph depicts the combined histological scores. Mean ± SD, n=3 (DSS), **p < 0.005, Student's unpaired t-test. Scale bar, 200 µm(E) The soluble and insoluble fractionations of lysates from colon tissues of Shigella-infected or DSS-treated Irgm1+/+ and Irgm-/- mice treated with GSK583 as indicated, were subjected to immunoblot analysis with indicated antibodies.(H-I) Western blot analysis with the colon tissue lysates of DSS-treated or S. flexneri infected mice groups as indicated.


Figure 1A ) Quantification of the effect of individual BCL - 2 - family proteins on HCT AKO 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 and * p < 0 , 05 with respect to GFP condition . C ) Western blot analysis of SMAC and cyt c release in GFP - BAX , tBID - GFP and GFP - BCLXL expressing cells at 12 h , 16 h and 24 h after transfection in HCT AKO . SN ( supernatant , cytosolic fraction ) , P ( pellet , membrane fraction ) and PS ( Ponceau Staining ) . 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 . 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 .

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Figure 1A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO 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 and * p<0,05 with respect to GFP condition.C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).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.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.


Figure 2A ) 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 . 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 . 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 .

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Figure 2A) 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.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.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.


Figure 3B ) 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 .

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Figure 3B) 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.


Figure 4A ) 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 .

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Figure 4A) 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.


Figure 5A ) 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 .

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Figure 5A) 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.


Figure 6A ) 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 . EH ) 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 .

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Figure 6A) 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.EH) 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.


Figure 1 ( 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

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Figure 1(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 loaded


Figure 2 ( 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

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Figure 2(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 μm


Figure 3 ( 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 μmRepresentative immunofluorescence images showed the expression of RPE - specific markers , including RPE65 ( B in the cultured RPE cells . Scale bars , 50 μmRepresentative 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 samples ( 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

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Figure 3(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 μmRepresentative immunofluorescence images showed the expression of RPE-specific markers, including RPE65 (B in the cultured RPE cells. Scale bars, 50 μmRepresentative 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 samples(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 fetus


Figure 4 ( 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 loaded ( 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

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Figure 4(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 loaded(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 μm


Figure 5 ( 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 μm ( 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

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Figure 5(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 μm(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 μm


Figure 1A . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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.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.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.


Figure 2A . 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 . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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.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).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.


Figure 3A . 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 . C . Time course of iodine - induced currents in hTRPA1 - expressing Xenopus oocytes . HC : HC030031D . 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 ) . 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 ) . I . Representative whole - cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin ( n = 3 ) .

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Figure 3A. 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.C. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. HC: HC030031D. 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).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).I. Representative whole-cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3).


Figure 1C . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C. 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.


Figure 2B . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. 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.


Figure 3A . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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.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.


Figure 4A . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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.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.


Figure 5C . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C. 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.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.


Figure 6C , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C, 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.


Figure 7B . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. 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 < 0.001


Figure 1Vector 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Vector 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.(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.(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.


Figure 2Vector 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Vector 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.(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.


Figure 3Vector 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Vector 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.(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


Figure 4Vector 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Vector 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.(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 layer


Figure 5Vector 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 5Vector 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.