( d - g ) Bioenergetics profiling of purified tumour cells from KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice ( e ) 6 weeks and ( g ) 18 weeks after tumour induction . Representative experimental data are shown . Oxygen consumption rates ( OCR ) were used to detect the bioenergetics profiles of purified tumour cells using a Seahorse Bioscience 24XF extracellular flux analyser . OCR ± s . e . m . were recorded to construct bioenergetics profiles for basal respiration ( BR ) , ATP turnover ( AT ) , respiratory capacity ( RC ) , spare respiratory capacity ( SRC ) and proton leakage ( PL ) . A minimum of seven different samples were analysed for each group . * P0 . 05 ; * * P0 . 01 ( unpaired , two - sided t - test ) .

O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d-g) Bioenergetics profiling of purified tumour cells from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice (e) 6 weeks and (g) 18 weeks after tumour induction. Representative experimental data are shown. Oxygen consumption rates (OCR) were used to detect the bioenergetics profiles of purified tumour cells using a Seahorse Bioscience 24XF extracellular flux analyser. OCR±s.e.m. were recorded to construct bioenergetics profiles for basal respiration (BR), ATP turnover (AT), respiratory capacity (RC), spare respiratory capacity (SRC) and proton leakage (PL). A minimum of seven different samples were analysed for each group. *P0.05; **P0.01(unpaired, two-sided t-test).


( a ) Increased tumour initiation in KRas ; Atg5fl / fl mice as compared with KRas ; Atg5fl / + littermates . Representative histological sections at 2 weeks after AdCre inhalation are shown . Insets show typical lung lesions . Haematoxylin and eosin staining . Scale bars , 2 mm for whole lung section ; 50 μm for insets .

O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O

(a) Increased tumour initiation in KRas;Atg5fl/fl mice as compared with KRas;Atg5fl/+ littermates. Representative histological sections at 2 weeks after AdCre inhalation are shown. Insets show typical lung lesions. Haematoxylin and eosin staining. Scale bars, 2 mm for whole lung section; 50 μm for insets.


( b ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 2 weeks after AdCre inhalation . n = 6 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) .

O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O O O O O O 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) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation. n=6 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link).


( c ) Tumour growth curves based on mean tumour - to - lung area ratios ( ± s . e . m . ) determined in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates at the indicated time points . At least three different planes from each lung were analysed . n ≥ 6 per cohort for each time point . * P0 . 05 , * * P0 . 01 ( unpaired , two - sided Wicoxon test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Tumour growth curves based on mean tumour-to-lung area ratios (±s.e.m.) determined in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates at the indicated time points. At least three different planes from each lung were analysed. n≥6 per cohort for each time point. *P0.05, **P0.01 (unpaired, two-sided Wicoxon test).


( d ) MicroCT analysis of lung tumours of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice 18 weeks after AdCre inhalation . Representative data from individual mice are shown . Scale bars , 5

O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O

(d) MicroCT analysis of lung tumours of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice 18 weeks after AdCre inhalation. Representative data from individual mice are shown. Scale bars, 5 mm


( a ) Distribution and ( b ) absolute numbers of hyperplastic lesions , adenomas and adenocarcinomas in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice6 , 12 and 18 weeks post AdCre infection . Data are shown as means ± s . e . m . ; n = 6 per group . Combining the tumour progression data from 6 , 12 and 18 weeks , a cumulative linked mixed model ( clmm ) analysis showed also a significant dependency of tumour progression on the genotype ; P0 . 05 ( χ2 - test of clmms assessing the genotype effect ) . Applying generalized linear - mixed effects ( glmer ) models with logit link to individual time points , shows a significant dependency of the transition from hyperplastic lesions to adenomas ( 12 weeks , P0 . 05 ) and from adenomas to adenocarcinomas ( 12 weeks , P0 . 01 and 18 weeks , P0 . 01 ) on the mouse genotype ( the last three P - values are from χ2 - tests of a generalized linear models with logit link assessing the genotype effect ) .

O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Distribution and (b) absolute numbers of hyperplastic lesions, adenomas and adenocarcinomas in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice6, 12 and 18 weeks post AdCre infection. Data are shown as means±s.e.m.; n=6 per group. Combining the tumour progression data from 6, 12 and 18 weeks, a cumulative linked mixed model (clmm) analysis showed also a significant dependency of tumour progression on the genotype; P0.05 (χ2-test of clmms assessing the genotype effect). Applying generalized linear-mixed effects (glmer) models with logit link to individual time points, shows a significant dependency of the transition from hyperplastic lesions to adenomas (12 weeks, P0.05) and from adenomas to adenocarcinomas (12 weeks, P0.01 and 18 weeks, P0.01) on the mouse genotype (the last three P-values are from χ2-tests of a generalized linear models with logit link assessing the genotype effect).


( c , d ) Histological analyses ( haematoxylin and eosin staining ) of lung tumours in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . ( c ) Nuclear and cellular polymorphy , more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 weeks after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours ( asterisk ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O

(c,d) Histological analyses (haematoxylin and eosin staining) of lung tumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).


( c , d ) Histological analyses ( haematoxylin and eosin staining ) of lungtumours in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . ( c ) Nuclear and cellular polymorphy , more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 weeks after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours ( asterisk ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O

(c,d) Histological analyses (haematoxylin and eosin staining) of lungtumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).


( a ) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot . β - actin is shown as a loading control .

O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(a) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot. β-actin is shown as a loading control.


( b ) Atg5 mRNA expression was determined by quantitative PCR . Data are shown as mean relative mRNA expression ± s . e . m . in KRas ; Atg5fl / fl tumours as compared with tumours from KRas ; Atg5fl / + mice ( set to 1 ) . Values were normalized to β - actin mRNA expression . n = 3 per group .

O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(b) Atg5 mRNA expression was determined by quantitative PCR. Data are shown as mean relative mRNA expression±s.e.m. in KRas;Atg5fl/fl tumours as compared with tumours from KRas;Atg5fl/+ mice (set to 1). Values were normalized to β-actin mRNA expression. n=3 per group.


( b ) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho - Chk2 in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . For γH2AX and phospho - Chk2 staining , sections were scored independently in a double - blinded manner . Data are shown as mean percentages ( ± s . e . m . ) of positive cells per total cell numbers . n = 6 mice per genotype . Scale bars , 50 μm . * * * P0 . 001 ( χ2 - test of a generalized linear model with logit link assessing the genotype effect ) .

O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho-Chk2 in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. For γH2AX and phospho-Chk2 staining, sections were scored independently in a double-blinded manner. Data are shown as mean percentages (±s.e.m.) of positive cells per total cell numbers. n=6 mice per genotype. Scale bars, 50 μm. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect).


( c ) Representative histological lung sections of KRas ; Atg5fl / + ; p53fl / fl and KRas ; Atg5fl / fl ; p53fl / fl littermate mice16 weeks after AdCre inhalation . Scale bars , 2 mm . ( d ) Distribution of hyperplastic lesions , adenomas and adenocarcinomas ( ± s . e . m . ) in KRas ; Atg5fl / + ; p53fl / fl and KRas ; Atg5fl / fl ; p53fl / fl littermates 16 weeks post AdCre infection ( n = 4 per genotype ) .

O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O

(c) Representative histological lung sections of KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermate mice16 weeks after AdCre inhalation. Scale bars, 2 mm. (d) Distribution of hyperplastic lesions, adenomas and adenocarcinomas (±s.e.m.) in KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermates 16 weeks post AdCre infection (n=4 per genotype).


( e ) The Kaplan - Meier survival curves for KRas ; Atg5fl / + ; p53fl / fl ( n = 8 ) and KRas ; Atg5fl / fl ; p53fl / fl ( n = 9 ) littermate mice injected intranasally with AdCre ( 2 . 5 × 107 p . f . u . ) . There was no statistical difference between the cohorts ( log - rank test ) .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) The Kaplan-Meier survival curves for KRas;Atg5fl/+;p53fl/fl (n=8) and KRas;Atg5fl/fl;p53fl/fl (n=9) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). There was no statistical difference between the cohorts (log-rank test).


( a , b ) Representative immunohistochemistry and quantitative assessment of ( a ) tumour - infiltrating CD3ε - positive T cells and ( b ) FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 6 weeks after AdCre inhalation . Data are shown as mean percentages ( ± s . e . m . ) of the indicated cell types per total cell numbers in the tumour areas . Only cells within tumours were scored . At least six mice per genotype were analysed . Scale bars , 50 μm . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) .

O O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O 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-TISSUE O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a,b) Representative immunohistochemistry and quantitative assessment of (a) tumour-infiltrating CD3ε-positive T cells and (b) FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 6 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of the indicated cell types per total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. ***P0.001(χ2-test assessing the genotype effect in a generalized linear model with logit link).


( c ) Representative immunohistochemistry and quantitative assessment of FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 2 weeks after AdCre inhalation . Data are shown as mean percentages ( ± s . e . m . ) of FoxP3 + cells among total cell numbers in the tumour areas . Only cells within tumours were scored . At least six mice per genotype were analysed . Scale bars , 50 μm . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) .

O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. **P0.01(χ2-test assessing the genotype effect in a generalized linear model with logit link).


( d ) FACS blots to detect CD25 + FoxP3 + Treg cells in lungs 2 weeks after AdCre inhalation . Cells were gated on CD4 + populations . Numbers indicate percentages among total CD4 + cells in the lungs .

O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-TISSUE O

(d) FACS blots to detect CD25+FoxP3+ Treg cells in lungs 2 weeks after AdCre inhalation. Cells were gated on CD4+ populations. Numbers indicate percentages among total CD4+ cells in the lungs.


( e ) Mediastinal lymph node and lung cells were isolated from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods . The left panel shows the percentage of CD4 + T cells among all lung - infiltrating CD45 + haematopoietic cells ( ± s . e . m . ) . The right panel shows the percentage of FoxP3 + cells among CD4 + T cells in mediastinal lymph nodes and lungs ( ± s . e . m . ) . n = 4 per genotype ; * P0 . 05 ( unpaired , two - sided t - test ) .

O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Mediastinal lymph node and lung cells were isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods. The left panel shows the percentage of CD4+ T cells among all lung-infiltrating CD45+ haematopoietic cells (±s.e.m.). The right panel shows the percentage of FoxP3+ cells among CD4+ T cells in mediastinal lymph nodes and lungs (±s.e.m.). n=4 per genotype; *P0.05 (unpaired, two-sided t-test).


( a ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti - CD25 Ab . n = 4 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( b ) Histological lung sections of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice treated with an isotype control Ab or anti - CD25 - immunodepleting Abs . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology . Representative lung images ( haematoxylin and eosin ( HE ) staining ) are shown for each genotype and treatment . Scale bars , 2 mm .

O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-TISSUE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-EXP_ASSAY O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti-CD25 Ab. n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). (b) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with an isotype control Ab or anti-CD25-immunodepleting Abs. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (haematoxylin and eosin (HE) staining) are shown for each genotype and treatment. Scale bars, 2 mm.


( c ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 2 weeks after AdCre inhalation treated either with an isotype control Ab or an Ab specific to FR4 . n = 4 per group . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( d ) Histological lung sections of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice treated with isotype control Abs or Abs specific to FR4 . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology . Representative lung images ( HE staining ) are shown for each genotype and treatment . Scale bars , 2 mm .

O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-TISSUE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-EXP_ASSAY O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with an isotype control Ab or an Ab specific to FR4. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). (d) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with isotype control Abs or Abs specific to FR4. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (HE staining) are shown for each genotype and treatment. Scale bars, 2 mm.


( b ) Protein expression of Hif1α , Atg5 and glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) in isolated KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes ( analysed at 4 days after AdCre infection ) and tumours purified from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice at week 2 after AdCre inhalation . Quantifications of the hypoxia - inducible factor ( HIF ) 1α / GAPDH ratios are shown for each lane ; two independent blots are quantified .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O B-GENEPROD O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

(b) Protein expression of Hif1α, Atg5 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes (analysed at 4 days after AdCre infection) and tumours purified from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice at week 2 after AdCre inhalation. Quantifications of the hypoxia-inducible factor (HIF)1α/GAPDH ratios are shown for each lane; two independent blots are quantified.


( c ) CD39 , Atg5 and as a control β - actin protein expression in isolated Atg5fl / + , Atg5fl / fl , KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes . Protein lysated were analysed at 4 days after AdCre infections by western blot . Quantifications of the CD39 / β - actin ratios are shown for each lane , normalized to the Atg5fl / + samples . Two independent blots are quantified .

O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(c) CD39, Atg5 and as a control β-actin protein expression in isolated Atg5fl/+, Atg5fl/fl, KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes. Protein lysated were analysed at 4 days after AdCre infections by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane, normalized to the Atg5fl/+ samples. Two independent blots are quantified.


( d , e ) CD39 , Atg5 and as a control β - actin protein expression in isolated KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes treated with the ( d ) HIF1α inhibitor 3 - ( 2 - ( 4 - adamantan - 1 - yl - phenoxy ) - acetylamino ) - 4 - hydroxybenzoic acid methyl ester or left untreated and ( e ) the Nrf2 inhibitor Brusatol ( 100 nM ) or the ROS scavenger N - acetyl - L - cysteine ( 5 mM ) . Protein lysates were analysed on day 4 by western blot . Quantifications of the CD39 / β - actin ratios are shown for each lane . The inhibitors were present during the entire 4 day culture period .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O

(d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4 day culture period.


( a ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) and ( b ) representative histological lung sections ( haematoxylin and eosin ( HE ) staining ) of KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 ( intraperitoneally ; i . p . 10 mg kg − 1 daily ) to inhibit CD39 activity . n = 4 per group . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . Scale bars , 2 mm .

O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (b) representative histological lung sections (haematoxylin and eosin (HE) staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 (intraperitoneally; i.p. 10 mg kg−1 daily) to inhibit CD39 activity. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.


( c ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) and ( d ) representative histological lung sections ( HE staining ) of KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 ( i . p . 10 mg kg − 1 daily ) . n = 4 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . Scale bars , 2 mm .

O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (d) representative histological lung sections (HE staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 (i.p. 10 mg kg−1 daily). n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.


( e , f ) Representative immunohistochemistry and quantitative assessment of FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates treated with vehicle control or the CD39 blocker polyoxometalate - 1 ( POM1 ) and treated with vehicle or the adenosine receptor inhibitor PSB1115 ( 4 - ( 2 , 3 , 6 , 7 - tetrahydro - 2 , 6 - dioxo - 1 - propyl - 1H - purin - 8 - yl ) - benzenesulphonic acid potassium salt ) . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology . Data are shown as mean percentages ( ± s . e . m . ) of FoxP3 + cells among total cell numbers in the tumour areas . Only cells within tumours were scored . At least four mice per genotype were analysed . Scale bars , 50 μm . * * P0 . 01 . NS , not significant ; ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) .

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e,f) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates treated with vehicle control or the CD39 blocker polyoxometalate-1 (POM1) and treated with vehicle or the adenosine receptor inhibitor PSB1115 (4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulphonic acid potassium salt). Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least four mice per genotype were analysed. Scale bars, 50 μm. **P0.01. NS, not significant; (χ2-test assessing the genotype effect in a generalized linear model with logit link).


( A ) Immunoblot with anti - TG2 or anti - β - actin as loading control in in whole lysates from small intestine homogenates of CftrF508del / F508del and wild - type ( CftrWT ) mice ( n = 5 per group ) and densitometric analysis of immunoblots . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 ( Student ' s t test ) . Data information : The blots are representative of one experiment for group of treatment .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O 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-EXP_ASSAY O O O O O O O O O O

(A) Immunoblot with anti-TG2 or anti-β-actin as loading control in in whole lysates from small intestine homogenates of CftrF508del/F508del and wild-type (CftrWT) mice (n=5 per group) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 (Student's t test). Data information: The blots are representative of one experiment for group of treatment.


( B ) Detection of NLRP3 expression ( top ) and caspase - 1 cleavage ( bottom ) by immunoblot of whole lysates from small intestine homogenates of CftrWT and CftrF508del / F508del mice ( n = 5 ) and densitometric analysis of immunoblots . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 CftrWT vs CftrF508del / F508del ( Student ' s t test ) . Data information : The blots are representative of one experiment for group of treatment .

O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

(B) Detection of NLRP3 expression (top) and caspase-1 cleavage (bottom) by immunoblot of whole lysates from small intestine homogenates of CftrWT and CftrF508del/F508del mice (n= 5) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrWT vs CftrF508del/F508del (Student's t test). Data information: The blots are representative of one experiment for group of treatment.


( C ) Protein levels of IL - 17A and IFN - γ from small intestine homogenates of CftrWT and CftrF508del / F508del ( n = 10 ) . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 CftrWT vs CftrF508del / F508del ( Student ' s t test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O

(C) Protein levels of IL-17A and IFN-γ from small intestine homogenates of CftrWT and CftrF508del/F508del (n= 10). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 CftrWT vs CftrF508del/F508del (Student's t test).


( D ) IL - 15 mRNA ( left ) and protein ( right ) levels in small intestine homogenates from CftrF508del / F508del , Cftr - / - or their CftrWT littermates ( n = 10 per group ) . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 CftrF508del / F508del vs CftrWT ( FVB / 129 ) , or ° ° p < 0 . 01 Cftr - / - vs CftrWT ( B6 . 129P2 ) ( ANOVA , Bonferroni post hoc test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O

(D) IL-15 mRNA (left) and protein (right) levels in small intestine homogenates from CftrF508del/F508del, Cftr-/- or their CftrWT littermates (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrF508del/F508del vs CftrWT(FVB/129), or °°p<0.01 Cftr-/- vs CftrWT(B6.129P2) (ANOVA, Bonferroni post hoc test).


( E ) IL - 15 mRNA levels in small intestine homogenates from CftrWT mice or CftrF508del / F508del or TG - / - / CftrF508del / F508del or TG - / - mice ( n = 10 per group ) . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 vs CftrWT , ° ° p < 0 . 01 , # # p < 0 . 01 vs CftrF508del / F508del ( ANOVA , Bonferroni post hoc test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O B-GENEPROD B-ORGANISM O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O 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-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(E) IL-15 mRNA levels in small intestine homogenates from CftrWT mice or CftrF508del/F508del or TG-/-/CftrF508del/F508del or TG-/- mice (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 vs CftrWT, °°p<0.01, ##p<0.01 vs CftrF508del/F508del (ANOVA, Bonferroni post hoc test).


( F ) Effects of 4 weeks of oral administration of gliadin on IL - 15 , IL - 17A and IFN - γ protein levels in small intestine homogenates from CftrF508del / F508del and CftrWT mice ( n = 10 mice per group of treatment ) . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 , * * * p < 0 . 001 ( CftrF508del / F508del vs CftrWT mice prior gliadin challenge ) , ° ° ° p < 0 . 001 ( CftrF508del / F508del mice vs CftrF508del / F508del mice after gliadin challenge ) , ( ANOVA , Bonferroni post hoc test ) .

O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O O O O O O O O O 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-GENEPROD O O O B-GENEPROD B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O

(F) Effects of 4 weeks of oral administration of gliadin on IL-15, IL-17A and IFN-γ protein levels in small intestine homogenates from CftrF508del/F508del and CftrWT mice (n =10 mice per group of treatment). Mean ± SD of triplicates of independent pooled samples. **p<0.01, ***p<0.001 (CftrF508del/F508del vs CftrWT mice prior gliadin challenge), °°°p<0.001 (CftrF508del/F508del mice vs CftrF508del/F508del mice after gliadin challenge), (ANOVA, Bonferroni post hoc test).


( G - I ) BALB / c mice ( G ) fed with a gluten - free diet for at least 3 generations , or ( H ) NOD or ( I ) NOD - DQ8 mice orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) . Representative traces of CFTR - dependent Cl - secretion measured by forskolin ( Fsk ) - induced increase of chloride current ( Isc ( μA / cm2 ) ) in small intestines mounted in Ussing chambers ; quantification of the peak CFTR inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in tissue samples ( n = 3 independent experiments ) . Mean ± SD of samples assayed ; * * p < 0 . 01 , * * * p < 0 . 001 vs challenged with gliadin ( Student ' s t test ) .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O 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-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O

(G-I) BALB/c mice (G) fed with a gluten-free diet for at least 3 generations, or (H) NOD or (I) NOD-DQ8 mice orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks). Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=3 independent experiments). Mean±SD of samples assayed; **p<0.01, ***p<0.001 vs challenged with gliadin (Student's t test).


( A ) Representative traces of CFTR - dependent Cl - secretion measured by forskolin ( Fsk ) - inducible chloride current ( Isc ( μA / cm2 ) ) in Caco - 2 cells mounted in Ussing chambers after 3 h of incubation with P57 - 68 or P33 - mer or P31 - 43 peptides ( 20 µg / ml ) and of P31 - 43 in the presence of up to 30 minutes of pre - treatment of VX - 770 ( 10 µM ) ; quantification of the peak CFTR Inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in Caco - 2 cells ( n = 3 independent experiments ) . Mean ± SD of samples assayed . * * * p < 0 . 001 vs P31 - 43 challenge , # # p < 0 . 01 vs Caco - 2 challenged with P31 - 43 in the presence of VX - 770 ( ANOVA , Bonferroni post hoc test ) .

O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-inducible chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers after 3 h of incubation with P57-68 or P33-mer or P31-43 peptides (20 µg/ml) and of P31-43 in the presence of up to 30 minutes of pre-treatment of VX-770 (10 µM); quantification of the peak CFTR Inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. ***p<0.001 vs P31-43 challenge, ##p<0.01 vs Caco-2 challenged with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).


( B ) Incubation of Caco - 2 cells with P31 - 43 or control peptides ( p57 - 68 or PGAV ) for 1 h that were preceded or not by 20 min of pre - treatment with VX - 770 . Immunoprecipitation in non - reducing and non - denaturing conditions of CFTR protein and immunblot with streptavidin - HRP or CFTR antibody . Data information : The blots are representative of one experiment for group of treatment .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O

(B) Incubation of Caco-2 cells with P31-43 or control peptides (p57-68 or PGAV) for 1 h that were preceded or not by 20 min of pre-treatment with VX-770. Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody. Data information: The blots are representative of one experiment for group of treatment.


( E ) Surface plasmon resonance ( SPR ) analysis of increasing concentrations of P31 - 43 and P57 - 68 peptides on rhNBD1 covalently bound to the CM5 sensor chip .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(E) Surface plasmon resonance (SPR) analysis of increasing concentrations of P31-43 and P57-68 peptides on rhNBD1 covalently bound to the CM5 sensor chip.


( F ) Surface plasmon resonance ( SPR ) analysis of rhNBD1 binding to P31 - 43 and P57 - 68 biotinylated peptides immobilized on SA sensor chip .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O O O O O O

(F) Surface plasmon resonance (SPR) analysis of rhNBD1 binding to P31-43 and P57-68 biotinylated peptides immobilized on SA sensor chip.


Blue native polyacrylamide gel electrophoresis ( PAGE ) western blotting of P31 - 43 and P57 - 68 biotinylated peptides in the presence of rhNBD1 ( G ) All the recombinant proteins and the indicated peptides were pre - incubated in an appropriate buffer at 4 ° C for 30 min and then resolved in native conditions to preserve the formation of peptide / protein complexes . Data information : The blots are representative of one experiment for group of treatment .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O

Blue native polyacrylamide gel electrophoresis (PAGE) western blotting of P31-43 and P57-68 biotinylated peptides in the presence of rhNBD1 (G) All the recombinant proteins and the indicated peptides were pre-incubated in an appropriate buffer at 4°C for 30 min and then resolved in native conditions to preserve the formation of peptide/protein complexes. Data information: The blots are representative of one experiment for group of treatment.


Blue native polyacrylamide gel electrophoresis ( PAGE ) western blotting of WT and double NBD1 mutants in the presence of biotinylated P31 - 43 ( H ) . All the recombinant proteins and the indicated peptides were pre - incubated in an appropriate buffer at 4 ° C for 30 min and then resolved in native conditions to preserve the formation of peptide / protein complexes . Data information : The blots are representative of one experiment for group of treatment .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O

Blue native polyacrylamide gel electrophoresis (PAGE) western blotting of WT and double NBD1 mutants in the presence of biotinylated P31-43 (H). All the recombinant proteins and the indicated peptides were pre-incubated in an appropriate buffer at 4°C for 30 min and then resolved in native conditions to preserve the formation of peptide/protein complexes. Data information: The blots are representative of one experiment for group of treatment.


( I ) P31 - 43 induced modifications on NBD1 ATP binding site using the intrinsic W401 fluorescence .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O

(I) P31-43 induced modifications on NBD1 ATP binding site using the intrinsic W401 fluorescence.


( J ) P31 - 43 effect on NBD1 ATPase activity .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O

(J) P31-43 effect on NBD1 ATPase activity.


( K ) P31 - 43 effect on NBD1 ATPase activity , applied at its IC50 , compared to that obtained with 5µM of the non - hydrolyzable ATP analogue ( P - ATP ) as a positive control of inhibition . Mean ± SD of triplicates of independent experiments .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(K) P31-43 effect on NBD1 ATPase activity, applied at its IC50, compared to that obtained with 5µM of the non-hydrolyzable ATP analogue (P-ATP) as a positive control of inhibition. Mean ± SD of triplicates of independent experiments.


( B ) SPR analysis of 4QA peptide on rhNBD1 covalently bound to CM5 sensor chip .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O

(B) SPR analysis of 4QA peptide on rhNBD1 covalently bound to CM5 sensor chip.


Incubation of Caco - 2 cells with P31 - 43 or with modified P31 - 43 ( P31 - 43 - 2QA 4 - 5th or P31 - 43 - 2QA 10 - 11th or P31 - 43 / 4QA mutant ( 4QA ) peptides ) for 1 h . ( C ) Immunoprecipitation in non - reducing and non - denaturing conditions of CFTR protein and immunblot with streptavidin - HRP or CFTR antibody ( n = 3 independent experiments ) . Mean ± SD of samples assayed . ° ° p < 0 . 01 vs P31 - 43 challenge , * * * p < 0 . 001 vs Caco - 2 challenged with 4QA ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Incubation of Caco-2 cells with P31-43 or with modified P31-43 (P31-43-2QA 4-5th or P31-43-2QA 10-11th or P31-43/4QA mutant (4QA) peptides) for 1 h. (C) Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody (n=3 independent experiments). Mean ± SD of samples assayed. °°p<0.01 vs P31-43 challenge, ***p<0.001 vs Caco-2 challenged with 4QA (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Incubation of Caco - 2 cells with P31 - 43 or with modified P31 - 43 ( P31 - 43 - 2QA 4 - 5th or P31 - 43 - 2QA 10 - 11th or P31 - 43 / 4QA mutant ( 4QA ) peptides ) for 1 h . ( D ) Representative traces of CFTR - dependent Cl - secretion measured by forskolin ( Fsk ) - induced increase of chloride current ( Isc ( μA / cm2 ) ) in Caco - 2 cells mounted in Ussing chambers ; quantification of the peak CFTR inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in Caco - 2 cells ( n = 3 independent experiments ) . Mean ± SD of samples assayed . ° ° p < 0 . 01 vs P31 - 43 challenge , * * * p < 0 . 001 vs Caco - 2 challenged with 4QA ( ANOVA , Bonferroni post hoc test ) .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O

Incubation of Caco-2 cells with P31-43 or with modified P31-43 (P31-43-2QA 4-5th or P31-43-2QA 10-11th or P31-43/4QA mutant (4QA) peptides) for 1 h. (D) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. °°p<0.01 vs P31-43 challenge, ***p<0.001 vs Caco-2 challenged with 4QA (ANOVA, Bonferroni post hoc test).


( E ) In vitro Blue native PAGE western blotting of enhanced binding of P31 - 43 to rhNBD1 in the presence of activated rTG2 that in turn promotes the formation of higher molecular weight complexes . As described in the text , only P31 - 43 ( lanes 1 , 3 , 5 ) , and not P57 - 68 ( lanes 2 , 4 , 6 ) , has the ability to form stable complexes with NBD1 and TG2 ( n = 3 independent experiments ) . Data information : The blots are representative of one experiment for group of treatment .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

(E) In vitro Blue native PAGE western blotting of enhanced binding of P31-43 to rhNBD1 in the presence of activated rTG2 that in turn promotes the formation of higher molecular weight complexes. As described in the text, only P31-43 (lanes 1, 3, 5), and not P57-68 (lanes 2, 4, 6), has the ability to form stable complexes with NBD1 and TG2 (n=3 independent experiments). Data information: The blots are representative of one experiment for group of treatment.


Incubation of Caco - 2 cells with P31 - 43 , in the presence or absence of pre - treatment with TG2 inhibitor Z - DON , TG2 - siRNA Immunoprecipitation in non - reducing and non - denaturing conditions of CFTR protein and immunoblot with anti - isopeptide glutamime - lysine and

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

Incubation of Caco-2 cells with P31-43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON, TG2-siRNA Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunoblot with anti-isopeptide glutamime-lysine and CFTR


Incubation of Caco - 2 cells with P31 - 43 in the presence or absence of pre - treatment with TG2 inhibitor Z - DON BAPTA - AM immunoprecipitation of CFTR and then immunoblot with anti - TG2 or anti - CFTR

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD

Incubation of Caco-2 cells with P31-43 in the presence or absence of pre-treatment with TG2 inhibitor Z-DON BAPTA-AM immunoprecipitation of CFTR and then immunoblot with anti-TG2 or anti-CFTR antibodies


Incubation of Caco - 2 cells with P31 - 43 , in the presence or absence of pre - treatment with TG2 inhibitor Z - DON or VX - 770 . Effects of treatment with VX - 770 and Z - DON on TG2 transamidating activity in situ . ( H ) In situ detection of TG2 activity in Caco - 2 cells pulsed with Ca2 + by immunoblotting of the TG - catalyzed incorporation of 5 - biotinamidopentylamine ( BAP ) and blotting with anti - biotin antibody ( n = 3 independent experiments ) . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 vs P31 - 43 , ° ° p < 0 . 001 vs VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Incubation of Caco-2 cells with P31-43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON or VX-770. Effects of treatment with VX-770 and Z-DON on TG2 transamidating activity in situ. (H) In situ detection of TG2 activity in Caco-2 cells pulsed with Ca2+ by immunoblotting of the TG-catalyzed incorporation of 5-biotinamidopentylamine (BAP) and blotting with anti-biotin antibody (n=3 independent experiments). Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Incubation of Caco - 2 cells with P31 - 43 , in the presence or absence of pre - treatment with TG2 inhibitor Z - DON VX - 770 . Effects of treatment with VX - 770 and Z - DON on TG2 transamidating activity in situ . ( I ) Assay of TG2 activity by immunostaining of the TG - catalysed incorporation of monodansylcadaverin in Caco - 2 cells pre - treated with VX - 770 or Z - DON and then pulsed with P31 - 43 . Scale bar , 50µm . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 vs P31 - 43 , ° ° p < 0 . 001 vs VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Incubation of Caco-2 cells with P31-43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON VX-770. Effects of treatment with VX-770 and Z-DON on TG2 transamidating activity in situ. (I) Assay of TG2 activity by immunostaining of the TG-catalysed incorporation of monodansylcadaverin in Caco-2 cells pre-treated with VX-770 or Z-DON and then pulsed with P31-43. Scale bar, 50µm. Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Incubation of Caco - 2 cells with P57 - 68 or P31 - 43 , in the presence or absence of pre - treatment with VX - 770 . ( J ) Immunoprecipitation of CFTR and immunoblot with anti - CFTR and anti - TG2 antibodies . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 vs P31 - 43 , ° ° p < 0 . 001 vs VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Incubation of Caco-2 cells with P57-68 or P31-43, in the presence or absence of pre-treatment with VX-770. (J) Immunoprecipitation of CFTR and immunoblot with anti-CFTR and anti-TG2 antibodies. Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Incubation of Caco - 2 cells with P57 - 68 or P31 - 43 , in the presence or absence of pre - treatment with VX - 770 . ( K ) Immunoprecipitation in non - reducing and non - denaturing conditions of PKAr2α protein and immunoblot with isopeptide glutamine - lysine and PKAr2α antibodies ( n = 3 independent experiments ) . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 vs P31 - 43 , ° ° p < 0 . 001 vs VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Incubation of Caco-2 cells with P57-68 or P31-43, in the presence or absence of pre-treatment with VX-770. (K) Immunoprecipitation in non-reducing and non-denaturing conditions of PKAr2α protein and immunoblot with isopeptide glutamine-lysine and PKAr2α antibodies (n=3 independent experiments). Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Incubation of Caco - 2 cells with P57 - 68 or P31 - 43 , in the presence or absence of pre - treatment with VX - 770 . ( L ) Immunoblot of phospho - PKA ( phPKA ) protein ; densitometric analysis of protein levels relative to β - actin . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 vs P31 - 43 , ° ° p < 0 . 001 vs VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Incubation of Caco-2 cells with P57-68 or P31-43, in the presence or absence of pre-treatment with VX-770. (L) Immunoblot of phospho-PKA (phPKA) protein; densitometric analysis of protein levels relative to β-actin. Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Caco - 2 cells incubated with P31 - 43 in the presence or absence of VX - 770 ( A ) Immunoblot of PPARγ or phospho - ERK1 / 2 ( phERK1 / 2 ) and densitometric analysis of protein levels relative to β - actin . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 vs P31 - 43 , ° ° p < 0 . 001 vs VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (A) Immunoblot of PPARγ or phospho-ERK1/2 (phERK1/2) and densitometric analysis of protein levels relative to β-actin. Mean ± SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Caco - 2 cells incubated with P57 - 68 or PGAV or with P31 - 43 in the presence or absence of VX - 770 Immunoblotting with specific antibodies in Caco - 2 cells challenged for 2 or 4 h in the presence or absence of VX - 770 . NF - κB p65 in cytoplasmic and nuclear extracts ( B ) Densitometric analysis of protein levels relative to loading control . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 and ° ° p < 0 . 001 vs 2h or 4h of culture with P31 - 43 in the presence of VX - 770 ( ANOVA , Bonferroni post hoc test ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

Caco-2 cells incubated with P57-68 or PGAV or with P31-43 in the presence or absence of VX-770 Immunoblotting with specific antibodies in Caco-2 cells challenged for 2 or 4 h in the presence or absence of VX-770. NF-κB p65 in cytoplasmic and nuclear extracts (B) Densitometric analysis of protein levels relative to loading control. Mean ± SD of triplicates of independent experiments. **p<0.01 and °°p<0.001 vs 2h or 4h of culture with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).


Caco - 2 cells incubated with P57 - 68 or PGAV or with P31 - 43 in the presence or absence of VX - 770 Immunoblotting with specific antibodies in Caco - 2 cells challenged for 2 or 4 h in the presence or absence of VX - 770 . NLRP3 expression ( C ) Densitometric analysis of protein levels relative to loading control . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 and ° ° p < 0 . 001 vs 2h or 4h of culture with P31 - 43 in the presence of VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Caco-2 cells incubated with P57-68 or PGAV or with P31-43 in the presence or absence of VX-770 Immunoblotting with specific antibodies in Caco-2 cells challenged for 2 or 4 h in the presence or absence of VX-770. NLRP3 expression (C) Densitometric analysis of protein levels relative to loading control. Mean ± SD of triplicates of independent experiments. **p<0.01 and °°p<0.001 vs 2h or 4h of culture with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Caco - 2 cells incubated with P57 - 68 or PGAV or with P31 - 43 in the presence or absence of VX - 770 Immunoblotting with specific antibodies in Caco - 2 cells challenged for 2 or 4 h in the presence or absence of VX - 770 . caspase - 1 cleavage ( D ) . Densitometric analysis of protein levels relative to loading control . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 and ° ° p < 0 . 001 vs 2h or 4h of culture with P31 - 43 in the presence of VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Caco-2 cells incubated with P57-68 or PGAV or with P31-43 in the presence or absence of VX-770 Immunoblotting with specific antibodies in Caco-2 cells challenged for 2 or 4 h in the presence or absence of VX-770. caspase-1 cleavage (D). Densitometric analysis of protein levels relative to loading control. Mean ± SD of triplicates of independent experiments. **p<0.01 and °°p<0.001 vs 2h or 4h of culture with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Caco - 2 cells incubated with P57 - 68 or with P31 - 43 in the presence or absence of VX - 770 or Vrx - 532 . ( E ) Immunoblot of membrane protein fractions with anti - NHERF - 1 , anti - ezrin Abs and anti - flotillin as a control . Densitometric analysis of immunoblot ( mean ± SD of triplicates of independent experiments ) ; * p < 0 . 01 vs treatment with VX - 770 or ° ° p < 0 . 01 vs treatment with Vrx - 532 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

Caco-2 cells incubated with P57-68 or with P31-43 in the presence or absence of VX-770 or Vrx-532. (E) Immunoblot of membrane protein fractions with anti-NHERF-1, anti-ezrin Abs and anti-flotillin as a control. Densitometric analysis of immunoblot (mean ± SD of triplicates of independent experiments); *p<0.01 vs treatment with VX-770 or °°p<0.01 vs treatment with Vrx-532 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.


Caco - 2 cells incubated with P31 - 43 in the presence or absence of VX - 770 ( F ) Confocal image staining with anti - F - actin . DAPI ( blue ) , nuclear counterstaining . Scale bar , 50 µm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O

Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (F) Confocal image staining with anti-F-actin. DAPI (blue), nuclear counterstaining. Scale bar, 50 µm.


Caco - 2 cells incubated with P31 - 43 in the presence or absence of VX - 770 ( G ) IL - 15 production ( quantified by specific ELISA ) in CFTR - WT Caco - 2 cells treated or not with P31 - 43 ( left ) or in Caco - 2CFTR - KO cells ( right ) , in the presence or absence of VX - 770 . Mean ± SD of triplicates of independent experiments . * * * p < 0 . 001 vs untreated and ° ° ° p < 0 . 001 vs CFTR - WT Caco - 2 cells treated with P31 - 43 + VX - 770 ( ANOVA , Bonferroni post hoc test ) . * * p < 0 . 01 vs Caco - 2 CFTR WT ( Student ' s t - test ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O 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-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O O O O O O O

Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (G) IL-15 production (quantified by specific ELISA) in CFTR-WT Caco-2 cells treated or not with P31-43 (left) or in Caco-2CFTR-KO cells (right), in the presence or absence of VX-770. Mean ± SD of triplicates of independent experiments. ***p<0.001 vs untreated and °°°p<0.001 vs CFTR-WT Caco-2 cells treated with P31-43+VX-770 (ANOVA, Bonferroni post hoc test). **p<0.01 vs Caco-2 CFTR WT (Student's t-test).


Caco - 2 cells incubated with P31 - 43 in the presence or absence of VX - 770 ( H ) Caco - 2 or Caco - 2CFTR - KO cells in the presence or absence of pre - treatment with VX - 770 . Immunoblot of phERK1 \ 2 or β - actin ( left ) ; densitometric analysis of protein levels ( right ) . Mean ± SD of triplicates of independent experiments . * p < 0 . 05 ( Student ' s t - test ) . Data information : The blots are representative of one experiment for group of treatment .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O

Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (H) Caco-2 or Caco-2CFTR-KO cells in the presence or absence of pre-treatment with VX-770. Immunoblot of phERK1\2 or β-actin (left); densitometric analysis of protein levels (right). Mean ± SD of triplicates of independent experiments. *p<0.05 (Student's t-test). Data information: The blots are representative of one experiment for group of treatment.


BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( A ) Representative traces of CFTR - dependent Cl - secretion measured by forskolin ( Fsk ) - induced increase of chloride current ( Isc ( μA / cm2 ) ) in small intestines mounted in Ussing chambers ; quantification of the peak CFTR inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in tissue samples ( n = 5 ) . Mean ± SD of samples assayed . * * * p < 0 . 001 gliadin vs gliadin + VX - 770 ( Student ' s t - test ) .

B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=5). Mean ± SD of samples assayed. ***p<0.001 gliadin vs gliadin+VX-770 (Student's t-test).


BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( B ) Immunoblot with anti - CFTR antibody and β - actin loading control . Densitometric analysis are mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 vehicle vs gliadin ; ° p < 0 . 05 , gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post test ) . Data information : The blots are representative of one experiment for group of treatment .

B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (B) Immunoblot with anti-CFTR antibody and β-actin loading control. Densitometric analysis are mean ± SD of triplicates of independent pooled samples. **p<0.01 vehicle vs gliadin; °p<0.05, gliadin vs VX-770+gliadin (ANOVA, Bonferroni post test). Data information: The blots are representative of one experiment for group of treatment.


BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( C ) Plasma markers of intestinal permeability in mice . Plasma concentration of FITC - dextran 4000 ( FITC - D4000 ) measured 1 h after gavage of a single dose of 600 mg FITCD4000 per kg body weight . Quantification of plasma concentration from n = 10 mice per group of treatment expressed as mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 vehicle vs gliadin ; ° ° ° p < 0 . 01 , gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post test ) .

B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O B-TISSUE B-EXP_ASSAY O B-ORGANISM O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE B-EXP_ASSAY O O O O B-ORGANISM O O 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-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (C) Plasma markers of intestinal permeability in mice. Plasma concentration of FITC-dextran 4000 (FITC-D4000) measured 1 h after gavage of a single dose of 600 mg FITCD4000 per kg body weight. Quantification of plasma concentration from n=10 mice per group of treatment expressed as mean ± SD of triplicates of independent pooled samples. ***p<0.001 vehicle vs gliadin; °°°p<0.01, gliadin vs VX-770+gliadin (ANOVA, Bonferroni post test).


BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( D ) NLRP3 expression by immunoblotting with specific antibodies in small intestines from 3 mice . Densitometric analysis are mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 vehicle vs gliadin ; ° p < 0 . 05 , gliadin vs VX - 770 + gliadin . Data information : The blots are representative of one experiment for group of treatment .

B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O

BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (D) NLRP3 expression by immunoblotting with specific antibodies in small intestines from 3 mice. Densitometric analysis are mean ± SD of triplicates of independent pooled samples. **p<0.01 vehicle vs gliadin; °p<0.05, gliadin vs VX-770+gliadin. Data information: The blots are representative of one experiment for group of treatment.


BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( E - F ) mRNA ( E ) or protein ( by specific ELISA ) ( F ) levels of IL - 15 , IL - 21 , IL - 17A and IFN - γ . Mean ± SD of triplicates of independent pooled samples . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vehicle vs gliadin ; ° p < 0 . 05 , ° ° p < 0 . 01 , ° ° ° p < 0 . 001 gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post hoc test ) .

B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O 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-SMALL_MOLECULE 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-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O

BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (E-F) mRNA (E) or protein (by specific ELISA) (F) levels of IL-15, IL-21, IL-17A and IFN-γ. Mean±SD of triplicates of independent pooled samples. *p<0.05, **p<0.01, ***p<0.001 vehicle vs gliadin; °p<0.05, °°p<0.01, °°°p<0.001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).


BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . Transcript level of Rorc and Tbet ( G ) from small intestine homogenates . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 or * * * * p < 0 . 0001 gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post hoc test ) .

B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O

BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). Transcript level of Rorc and Tbet (G) from small intestine homogenates. Mean ± SD of triplicates of independent pooled samples. ***p<0.001 or ****p<0.0001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).


BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . protein levels ( by specific ELISA ) of IL - 10 and TGF - β ( H ) from small intestine homogenates . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 or * * * * p < 0 . 0001 gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post hoc test ) .

B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O

BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). protein levels (by specific ELISA) of IL-10 and TGF-β (H) from small intestine homogenates. Mean ± SD of triplicates of independent pooled samples. ***p<0.001 or ****p<0.0001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).


IFN - γ release ( ELISA ) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co - culture model upon 24 h challenge of confluent CaCo - 2 cells in the upper compartment with PT - gliadin ( I ) in presence or absence of VX - 770 . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 , PT - gliadin vs medium ; ° ° ° p < 0 . 001 , PT - gliadin vs PT - gliadin + VX - 770 ( n = 4 ) ( ANOVA , Bonferroni post hoc test ) .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O

IFN-γ release (ELISA) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co-culture model upon 24 h challenge of confluent CaCo-2 cells in the upper compartment with PT-gliadin (I) in presence or absence of VX-770. Mean ± SD of triplicates of independent pooled samples. ***p<0.001, PT-gliadin vs medium; °°°p<0.001, PT-gliadin vs PT-gliadin + VX-770 (n=4) (ANOVA, Bonferroni post hoc test).


IFN - γ release ( ELISA ) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co - culture model upon 24 h challenge of confluent CaCo - 2 cells in the upper compartment with combination of P31 - 43 and P57 - 68 ( J ) in presence or absence of VX - 770 . Mean ± SD of triplicates of independent pooled samples . ; * * * p < 0 . 001 , P57 - 68 or P31 - 43 vs P31 - 43 / P57 - 68 combination ( n = 6 ) ; ° ° ° p < 0 . 001 , P57 - 68 / P31 - 43 combination vs VX - 770 + P57 - 68 / P31 - 43 ( n = 3 ) , ( ANOVA , Bonferroni post hoc test ) .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

IFN-γ release (ELISA) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co-culture model upon 24 h challenge of confluent CaCo-2 cells in the upper compartment with combination of P31-43 and P57-68 (J) in presence or absence of VX-770. Mean ± SD of triplicates of independent pooled samples. ; ***p<0.001, P57-68 or P31-43 vs P31-43/P57-68 combination (n=6); °°°p<0.001, P57-68/P31-43 combination vs VX-770 + P57-68/P31-43 (n=3), (ANOVA, Bonferroni post hoc test).


( K ) IL - 10 release ( ELISA ) in culture supernatants by PBMC from 4 celiac patients cultured as in ( J ) . Mean ± SD of triplicates of independent pooled samples . * * * * p < 0 . 0001 , P57 - 68 / P31 - 43 combination vs P57 - 68 / P31 - 43 + VX - 770 treatment ( n = 3 ) ( ANOVA , Bonferroni post hoc test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-DISEASE B-ORGANISM O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O

(K) IL-10 release (ELISA) in culture supernatants by PBMC from 4 celiac patients cultured as in (J). Mean ± SD of triplicates of independent pooled samples. ****p<0.0001, P57-68/P31-43 combination vs P57-68/P31-43+VX-770 treatment (n=3) (ANOVA, Bonferroni post hoc test).


( L ) IFN - γ release ( ELISA ) into culture supernatants by PBMC from 3 celiac patients cultured in the lower compartment of a bidimensional co - culture model upon a 24 h challenge of confluent Caco - 2CFTR - KO cells in the upper compartment with a combination of P31 - 43 and P57 - 68 in the presence or absence of VX - 770 , as in J and K . * p < 0 . 05 , medium vs P57 - 68 / P31 - 43 combination ( n = 3 ) ( ANOVA , Bonferroni post hoc test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

(L) IFN-γ release (ELISA) into culture supernatants by PBMC from 3 celiac patients cultured in the lower compartment of a bidimensional co-culture model upon a 24 h challenge of confluent Caco-2CFTR-KO cells in the upper compartment with a combination of P31-43 and P57-68 in the presence or absence of VX-770, as in J and K. *p<0.05, medium vs P57-68/P31-43 combination (n=3) (ANOVA, Bonferroni post hoc test).


( A - B ) Expression patterns ( blue stain ) of GUS reporter genes driven by the SKM1 ( A ) or SKM2 ( B ) promoter in roots of 5 - day - old Col - 0seedlings .

O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O

(A-B) Expression patterns (blue stain) of GUS reporter genes driven by the SKM1(A) or SKM2(B) promoter in roots of 5-day-old Col-0seedlings.


( C ) Non - complementation of CLE45 insensitivity of the bam3mutant by BAM3expressed under control of the SKM1promoter ( 7 - day - old seedlings ; 2 independent transgenic lines per construct are shown ) .

O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O

(C) Non-complementation of CLE45 insensitivity of the bam3mutant by BAM3expressed under control of the SKM1promoter (7-day-old seedlings; 2 independent transgenic lines per construct are shown).


( D ) Full CLE45 sensitivity of skm1or skm2 single and double mutant roots ( 7 - day - old seedlings ) .

O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O B-ORGANISM O O

(D) Full CLE45 sensitivity of skm1or skm2 single and double mutant roots (7-day-old seedlings).