( c ) Tumour growth curves on mean tumour - to - lung ratios ( ± s . e . m . ) determined in KRas ; Atg5fl / + KRas ; Atg5fl / fl at 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 ) .

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

(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 of lung tumours of KRas ; Atg5fl / + KRas ; Atg5fl / fl littermate mice 18 after AdCre inhalation . Representative data from mice are shown . Scale bars , 5

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

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 eosin ) of lung tumours in KRas ; Atg5fl / + KRas ; Atg5fl / fl 18 after AdCre inhalation . ( c ) polymorphy , more dense sometimes irregular as well as more nucleoli in tumour of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures necrotic areas in centre of larger tumours ( asterisk ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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 eosin ) of lungtumours in KRas ; Atg5fl / + KRas ; Atg5fl / fl 18 after AdCre inhalation . ( c ) polymorphy , more dense sometimes irregular as well as more nucleoli in tumour of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures necrotic areas in centre of larger tumours ( asterisk ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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 ) pneumocytes were infected with AdCre for 96 h Atg5 expression determined by western blot . β - actin is shown as a loading control .

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

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

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 KRas ; Atg5fl / fl ; p53fl / fl littermate mice16 after AdCre inhalation . Scale bars , 2 mm . ( d ) Distribution of hyperplastic lesions , adenomas adenocarcinomas ( ± s . e . m . ) in KRas ; Atg5fl / + ; p53fl / fl KRas ; Atg5fl / fl ; p53fl / fl 16 post AdCre infection ( n = 4 per genotype ) .

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

(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 ) Kaplan - Meier survival curves for KRas ; Atg5fl / + ; p53fl / fl ( n = 8 ) 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 cohorts ( log - rank ) .

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

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

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

O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 in lungs 2 after AdCre inhalation . were gated on CD4 + populations . Numbers indicate percentages among total CD4 + in lungs .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O 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 lung were isolated from KRas ; Atg5fl / + KRas ; Atg5fl / fl 2 after AdCre inhalation stained for flow cytometric as described in Methods . left panel shows of CD4 + T among lung - infiltrating CD45 + haematopoietic ( ± s . e . m . ) . right panel shows of FoxP3 + among CD4 + T in mediastinal lymph nodes lungs ( ± s . e . m . ) . n = 4 per genotype ; * P0 . 05 ( unpaired , two - sided t - ) .

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

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

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

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

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

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

O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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 as a control β - actin expression in isolated Atg5fl / + , Atg5fl / fl , KRas ; Atg5fl / + KRas ; Atg5fl / fl pneumocytes . lysated were analysed at 4 days after AdCre infections by western blot . Quantifications of CD39 / β - actin ratios are shown for each lane , normalized to Atg5fl / + samples . Two independent blots are quantified .

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

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

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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 ) of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) ( b ) representative histological lung sections ( haematoxylin eosin ( HE ) ) of KRas ; Atg5fl / + KRas ; Atg5fl / fl mice2 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 - assessing genotype effect in a generalized linear model with log link ) . Scale bars , 2 mm .

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

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

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

(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 quantitative assessment of FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + KRas ; Atg5fl / fl treated with vehicle control or CD39 blocker polyoxometalate - 1 ( POM1 ) treated with vehicle or 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 after AdCre inhalation their lungs were processed for immunohistopathology . Data are shown as mean percentages ( ± s . e . m . ) of FoxP3 + among total numbers in tumour areas . Only within tumours were scored . At least four mice per genotype were analysed . Scale bars , 50 μm . * * P0 . 01 . NS , not significant ; ( χ2 - assessing genotype effect in a generalized linear model with logit link ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 wild - type ( CftrWT ) mice ( n = 5 per group ) densitometric of immunoblots . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 ( Student ' s t ) . Data information : blots are representative of one experiment for group of treatment .

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

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

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

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 ) ( right ) in small intestine homogenates from CftrF508del / F508del , Cftr - / - or their CftrWT ( 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 ) .

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

(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 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 ) .

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

(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 of oral administration of gliadin on IL - 15 , IL - 17A IFN - γ in small intestine homogenates from CftrF508del / F508del 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 ) .

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

(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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) . 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 ; of 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 ) .

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

(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 mounted in Ussing chambers after 3 h of incubation with P57 - 68 or P33 - mer or P31 - 43 peptides ( 20 µg / ml ) of P31 - 43 in presence of up to 30 minutes of pre - treatment of VX - 770 ( 10 µM ) ; of peak CFTR Inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in Caco - 2 ( 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 presence of VX - 770 ( ANOVA , Bonferroni post hoc ) .

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

(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 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 non - denaturing conditions of CFTR immunblot with streptavidin - HRP or CFTR antibody . Data information : blots are representative of one experiment for group of treatment .

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

(B) 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 ) of increasing concentrations of P31 - 43 P57 - 68 peptides on rhNBD1 covalently bound to CM5 sensor chip .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O 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 ) of rhNBD1 binding to P31 - 43 P57 - 68 biotinylated peptides immobilized on SA sensor chip .

O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O 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 P57 - 68 biotinylated peptides in presence of rhNBD1 ( G ) recombinant indicated peptides were pre - incubated in an appropriate buffer at 4 ° C for 30 min then resolved in native conditions to preserve formation of peptide / complexes . Data information : blots are representative of one experiment for group of treatment .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 double NBD1 mutants in presence of biotinylated P31 - 43 ( H ) . recombinant indicated peptides were pre - incubated in an appropriate buffer at 4 ° C for 30 min then resolved in native conditions to preserve formation of peptide / complexes . Data information : blots are representative of one experiment for group of treatment .

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

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 intrinsic W401 .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O 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 non - hydrolyzable ATP analogue ( P - ATP ) as a positive control of inhibition . Mean ± SD of triplicates of independent experiments .

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

(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 of 4QA peptide on rhNBD1 covalently bound to CM5 sensor chip .

O O O O O B-MEASURED_VAR O O B-MEASURED_VAR 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 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 non - denaturing conditions of CFTR 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 ) . Data information : blots are representative of one experiment for group of treatment .

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

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 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 mounted in Ussing chambers ; of peak CFTR inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in Caco - 2 ( 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 ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 presence of activated rTG2 that in turn promotes formation of higher molecular complexes . As described in text , only P31 - 43 ( lanes 1 , 3 , 5 ) , not P57 - 68 ( lanes 2 , 4 , 6 ) , has ability to form stable complexes with NBD1 TG2 ( n = 3 independent experiments ) . Data information : blots are representative of one experiment for group of treatment .

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

(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 with P31 - 43 , in presence or absence of pre - treatment with TG2 inhibitor Z - DON , TG2 - siRNA Immunoprecipitation in non - reducing non - denaturing conditions of CFTR immunoblot with anti - isopeptide glutamime - lysine

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR 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, 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 with P31 - 43 in presence or absence of pre - treatment with TG2 inhibitor Z - DON BAPTA - AM immunoprecipitation of CFTR then immunoblot with anti - TG2 or anti - CFTR

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

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 with P31 - 43 , in presence or absence of pre - treatment with TG2 inhibitor Z - DON or VX - 770 . Effects of treatment with VX - 770 Z - DON on TG2 transamidating activity in situ . ( H ) In situ detection of TG2 activity in Caco - 2 pulsed with Ca2 + by immunoblotting of TG - catalyzed incorporation of 5 - biotinamidopentylamine ( BAP ) 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 ) . Data information : blots are representative of one experiment for group of treatment .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with P31 - 43 , in presence or absence of pre - treatment with TG2 inhibitor Z - DON VX - 770 . Effects of treatment with VX - 770 Z - DON on TG2 transamidating activity in situ . ( I ) of TG2 activity by immunostaining of TG - catalysed incorporation of monodansylcadaverin in Caco - 2 pre - treated with VX - 770 or Z - DON 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 ) . Data information : blots are representative of one experiment for group of treatment .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with P57 - 68 or P31 - 43 , in presence or absence of pre - treatment with VX - 770 . ( J ) Immunoprecipitation of CFTR immunoblot with anti - CFTR 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 ) . Data information : blots are representative of one experiment for group of treatment .

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

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 with P57 - 68 or P31 - 43 , in presence or absence of pre - treatment with VX - 770 . ( K ) Immunoprecipitation in non - reducing non - denaturing conditions of PKAr2α immunoblot with isopeptide glutamine - lysine 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 ) . Data information : blots are representative of one experiment for group of treatment .

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

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 with P57 - 68 or P31 - 43 , in presence or absence of pre - treatment with VX - 770 . ( L ) Immunoblot of phospho - PKA ( phPKA ) ; densitometric of 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 ) . Data information : blots are representative of one experiment for group of treatment .

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

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 incubated with P31 - 43 in presence or absence of VX - 770 ( A ) Immunoblot of PPARγ or phospho - ERK1 / 2 ( phERK1 / 2 ) densitometric of 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 ) . Data information : blots are representative of one experiment for group of treatment .

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

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

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

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

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

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

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 incubated with P57 - 68 or with P31 - 43 in presence or absence of VX - 770 or Vrx - 532 . ( E ) Immunoblot of fractions with anti - NHERF - 1 , anti - ezrin Abs anti - flotillin as a control . Densitometric 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 ) . Data information : blots are representative of one experiment for group of treatment .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 incubated with P31 - 43 in presence or absence of VX - 770 ( F ) Confocal image with anti - F - actin . DAPI ( blue ) , counterstaining . Scale bar , 50 µm .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) in 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 ; of 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 - ) .

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

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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) in 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 β - actin loading control . Densitometric 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 ) . Data information : blots are representative of one experiment for group of treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) in 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 of FITC - dextran 4000 ( FITC - D4000 ) measured 1 h after gavage of a single dose of 600 mg FITCD4000 per kg . of plasma 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). (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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) in 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 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 : blots are representative of one experiment for group of treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) in 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 ( by specific ELISA ) ( F ) of IL - 15 , IL - 21 , IL - 17A 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). (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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) in presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . Transcript level of Rorc 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 ( 5 mg / daily for one week then 5 mg / daily thrice a week for 3 ) in presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( by specific ELISA ) of IL - 10 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 ) .

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

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 or 4 controls cultured in lower compartment of a bidimensional co - culture model upon 24 h challenge of confluent CaCo - 2 in 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 ) .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 or 4 controls cultured in lower compartment of a bidimensional co - culture model upon 24 h challenge of confluent CaCo - 2 in upper compartment with combination of P31 - 43 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 ) .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 ) .

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

(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 cultured in lower compartment of a bidimensional co - culture model upon a 24 h challenge of confluent Caco - 2CFTR - KO in upper compartment with a combination of P31 - 43 P57 - 68 in presence or absence of VX - 770 , as in J K . * p < 0 . 05 , medium vs P57 - 68 / P31 - 43 combination ( n = 3 ) ( ANOVA , Bonferroni post hoc ) .

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

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

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

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

(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 double mutant roots ( 7 - day - old seedlings ) .

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

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


( E ) Failure of SKM1 or SKM2 expressed under control of BAM3 promoter to complement CLE45 insensitivity of bam3 mutant ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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

(E) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( A ) Root of 7 - day - old seedlings of indicated genotypes . ( B ) root of 7 - day - old seedlings . ( C ) Root meristem in 5 - day - old seedlings .

O O O O O O O O O O O O O O O O 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) Root phenotypes of 7-day-old seedlings of indicated genotypes. (B) Primary root length of 7-day-old seedlings. (C) Root meristem size in 5-day-old seedlings.


( D ) Root meristem phenotype of 5 - day - old seedlings , confocal microscopy , propidium iodide - stained ( red ) .

O 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) Root meristem phenotype of 5-day-old seedlings, confocal microscopy, propidium iodide-stained (red).


( E ) Auxin activity as visualized by constitutively expressed inverse DII - NLS - VENUS marker ( green ) in root meristems of 5 - day - old seedlings .

O O O O O O O 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) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings.


( F ) Gap frequency in brxsingle or brxmakr5 double mutants ( note : gap were absent from Col - 0 wild type or makr5single mutants ) . ( G ) Illustration of gap ( arrowheads ) in developing protophloem sieve element strands ( asterisks ) of brxsingle or brxmakr5 double mutants .

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

(F) Gap cell frequency in brxsingle or brxmakr5 double mutants (note: gap cells were absent from Col-0 wild type or makr5single mutants). (G) Illustration of gap cells (arrowheads) in developing protophloem sieve element strands (asterisks) of brxsingle or brxmakr5 double mutants.


( A ) root of 7 - day - old seedlings of indicated genotypes in response to continuous CLE45 treatments .

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

(A) Primary root length of 7-day-old seedlings of indicated genotypes in response to continuous CLE45 treatments.


( B ) Root meristem of ( CLE45 - treated ) 5 - day - old seedlings ( asterisks : protophloem sieve element strands ) . Note absence of protophloem in CLE45 - treated Col - 0 wild type roots .

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

(B) Root meristem phenotypes of (CLE45-treated) 5-day-old seedlings (asterisks: protophloem sieve element strands). Note absence of protophloem in CLE45-treated Col-0 wild type roots.


( C ) Transverse sections of ( CLE45 - treated ) 5 - day - old roots , just before onset of protoxylem ( asterisks ) differentiation . Note failure of protophloem sieve elements ( arrowheads ) to differentiate in CLE45 - treated Col - 0 ( indicated by dense cytoplasm ) .

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

(C) Transverse sections of (CLE45-treated) 5-day-old roots, just before the onset of protoxylem (asterisks) differentiation. Note failure of protophloem sieve elements (arrowheads) to differentiate in CLE45-treated Col-0 (indicated by dense cytoplasm).


( D ) Expression pattern of GUS reporter gene driven by MAKR5 promoter in roots of 5 - day - old Col - 0seedlings . ( E ) Corresponding transverse sections from different positions along root meristem .

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

(D) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings. (E) Corresponding transverse sections from different positions along the root meristem.


( F ) Expression of a MAKR5 - GFP fusion under control of MAKR5 promoter in roots of 5 - day - old seedlings .

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

(F) Expression of a MAKR5-GFP fusion protein under control of the MAKR5 promoter in roots of 5-day-old seedlings.


( G ) Corresponding optical sections illustrating MAKR5 - GFP expression in developing protophloem sieve elements ( asterisk ) adjacent files .

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

(G) Corresponding optical sections illustrating MAKR5-GFP expression in developing protophloem sieve elements (asterisk) and adjacent cell files.


( H ) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements ( CVP2 ) , but not by expression under control of a companion - specific promoter ( SUC2 ) ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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

(H) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements (CVP2), but not by expression under control of a companion cell-specific promoter (SUC2) (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( I ) Subcellular localization of MAKR5 - GFP ( meristematic zone ) . Note plasma association ( , green arrowheads ) dissociation from wall ( red arrowhead ) upon osmosis .

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

(I) Subcellular localization of MAKR5-GFP (meristematic zone). Note plasma membrane association (white, green arrowheads) and dissociation from the cell wall (red arrowhead) upon osmosis.


( B ) Expression of truncated MAKR5 fusion under control of MAKR5 promoter in 5 - day - old roots ( asterisks : protophloem sieve element strands ) .

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

(B) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5-day-old roots (asterisks: protophloem sieve element strands).


( C ) Close up of protophloem sieve element strands expressing truncated MAKR5 - CITRINE fusion under control of UBQ10 promoter .

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

(C) Close up of protophloem sieve element strands expressing truncated MAKR5-CITRINE fusion proteins under control of the UBQ10 promoter.


( D ) Failure of truncated MAKR5 fusion to complement CLE45 insensitivity of makr5 mutant ( 7 - day - old seedlings ; 1 - 2 independent transgenic lines per construct are shown ) .

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

(D) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant (7-day-old seedlings; 1-2 independent transgenic lines per construct are shown).


( E ) Sensitivity of makr5 mutant roots to continuous brassinolide ( BL ) treatment ( 7 - day - old seedlings ) .

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

(E) Sensitivity of makr5 mutant roots to continuous brassinolide (BL) treatment (7-day-old seedlings).


( F ) Expression of MAKR5 - BKI1 fusion under control of MAKR5 promoter in roots of 5 - day - old makr5 seedlings .

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

(F) Expression of MAKR5-BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5-day-old makr5 seedlings.


( G ) Complementation of CLE45 insensitivity of makr5 roots by BKI1 - MAKR5 , but not MAKR5 - BKI1 fusion ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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

(G) Complementation of CLE45 insensitivity of makr5 roots by BKI1-MAKR5, but not MAKR5-BKI1 fusion protein (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( A ) Increased expression of BAM3 - CITRINE fusion under control of BAM3 promoter in developing protophloem sieve elements ( asterisks ) of 5 - day - old bam3 roots upon CLE45 treatment .

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

(A) Increased expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements (asterisks) of 5-day-old bam3 roots upon CLE45 treatment.


( B ) Expression of BAM3 - CITRINE fusion under control of BAM3 promoter in 5 - day - old Col - 0 roots or makr5 roots .

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

(B) Expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots.


( C ) Expression of MAKR5 - GFP fusion under control of MAKR5 promoter in 5 - day - old Col - 0 or bam3 roots .

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

(C) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old Col-0 or bam3 roots.


( D ) GUS reporter gene expression driven by MAKR5 promoter in roots of 5 - day - old Col - 0seedlings upon CLE45 treatment .

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

(D) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings upon CLE45 treatment.


( E ) Expression of MAKR5 - GFP fusion under control of MAKR5 promoter in 5 - day - old roots upon CLE45 treatment ( 3 h ) . Note accumulation of MAKR5 - GFP in protophloem sieve elements ( magnified ) its dependence on functional BAM3 CLE45 . frequency of representative observations per total observations is indicated .

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

(E) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old roots upon CLE45 treatment (3 h). Note accumulation of MAKR5-GFP in protophloem sieve elements (magnified) and its dependence on functional BAM3 and CLE45. The frequency of representative observations per total observations is indicated.


( F ) Close up view of MAKR5 - GFP accumulation in protophloem file upon CLE45 treatment .

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

(F) Close up view of MAKR5-GFP accumulation in the protophloem cell file upon CLE45 treatment.


( G - H ) Accumulation of MAKR5 - BKI1 fusion in protophloem upon CLE45 treatment . frequency of representative observations per total observations is indicated . ( I - J ) of fusion abundance in developing protophloem upon expression under control of MAKR5 promoter , showing mean total ( I ) plasma to cytosol signal ( J ) .

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

(G-H) Accumulation of MAKR5-BKI1 fusion proteins in protophloem cells upon CLE45 treatment. The frequency of representative observations per total observations is indicated. (I-J) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter, showing mean total fluorescence (I) and plasma membrane to cytosol signal ratio (J).


B . Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs .

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

B. Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs.


C . qPCR validations of neutrophil genes . n = 7 DFUs n = 3 Skin D3 wounds . * * P < 0 . 01 ( two - unpaired Student ' s t - ) . Data presented as mean ± SD .

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C. qPCR validations of neutrophil genes. n=7 DFUs and n=3 Skin D3 wounds. **P < 0.01 (two-tailed unpaired Student's t-test). Data presented as mean ± SD.


A . Representative of human neutrophils treated with FOXM1 inhibitor , FDI - 6 . Vehicle ( DMSO ) served as a control . Neutrophils undergoing NET formation are visualized in green live neutrophils are visualized in red . was performed by normalizing number of neutrophils undergoing NET formation to number of live neutrophils . Neutrophils from n = 3 different blood donors were isolated , pooled peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc ) . Data presented as mean ± SD . ( Scale bar : 100µm ) .

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A. Representative images of human neutrophils treated with the FOXM1 inhibitor, FDI-6. Vehicle (DMSO) served as a control. Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red. Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). Data presented as mean ± SD. (Scale bar: 100µm).


B . of ROS in human neutrophils treated with FDI - 6 or in combination with N - acetylcysteine ( NAC ) . Phorbol 12 - myristate 13 - acetate ( PMA ) treatment served as a positive control . Data presented as mean ± SD . Neutrophils from n = 3 different blood donors were isolated , pooled peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc ) . C . of NET formation in human neutrophils treated with FDI - 6 or in combination with NAC . PMA treatment served as a positive control . Neutrophils from n = 3 different blood donors were isolated , pooled peformed in triplicate . Data presented as mean ± SD . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc ) .

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B. Quantification of ROS levels in human neutrophils treated with FDI-6 or in combination with N-acetylcysteine (NAC). Phorbol 12-myristate 13-acetate (PMA) treatment served as a positive control. Data presented as mean ± SD. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). C. Quantification of NET formation in human neutrophils treated with FDI-6 or in combination with NAC. PMA treatment served as a positive control. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. Data presented as mean ± SD. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).


A . Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response .

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A. Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response.


A . Representative of wounded skin after topical treatment with either vehicle ( IgG isotype control ) or ⍺ - TREM1 activator at 0 , 2 , 4 , 6 , 8 days after wounding . of wound at each time following vehicle or ⍺ - TREM1 activator treatment relative to original wound . of wound areas in n = 10 for vehicle diabetic 12 wounds for ⍺ - TREM1 treated wounds were performed with Fiji software . Data presented as mean ± SD . * * P < 0 . 01 , * * * * P < 0 . 0001 ( two - way ANOVA followed by Tukey ' s post - hoc ) .

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

A. Representative images of wounded skin after topical treatment with either vehicle (IgG isotype control) or ⍺-TREM1 activator at 0, 2, 4, 6, and 8 days after wounding. Percent of wound area at each time following vehicle or ⍺-TREM1 activator treatment relative to the original wound area. Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺-TREM1 treated wounds were performed with Fiji software. Data presented as mean ± SD. **P < 0.01, and ****P < 0.0001 (two-way ANOVA followed by Tukey's post-hoc test).