E . Silique pictures of cycb1 double mutant combinations . arrowheads indicate aborted ovules seeds . Scale bars : 500 μm . F , G . of aborted seeds in single ( F ) double ( G ) mutants . Graphs represents average seed abortion rate per ± SD of three biological replicates , n = 550 - 1029 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA , followed by a Dunnett ' s multiple comparisons ( * * * * P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Silique pictures of cycb1 double mutant combinations. White arrowheads indicate aborted ovules and seeds. Scale bars: 500 μm. F, G. Quantification of aborted seeds in single (F) and double (G) mutants. Graphs represents the average seed abortion rate per plant ± SD of three biological replicates, n = 550-1029 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


C , D . of endosperm nuclei in cycb1 single ( C ) double ( D ) mutants . Boxes whiskers represent min to max values with median indicated as a central horizontal , n = 26 - 30 seeds per genotype . Asterisks show significant differences in number of endosperm nuclei per seed in a Kruskal - Wallis followed by Dunn ' s multiple comparisons ( * * * * P < 0 . 0001 ) .

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

C, D. Quantification of endosperm nuclei in cycb1 single (C) and double (D) mutants. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 26-30 seeds per genotype. Asterisks show significant differences in the number of endosperm nuclei per seed in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (**** P < 0.0001).


A . DIC of abnormal sacs in cycb1 mutant combinations . Red arrowheads indicate visible nuclei in Col - 0 sacs ( central egg ) corresponding structures in quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant . Scale bars : 20 μm . B . of different abnormal sac structures in cycb1 mutant combinations ( n = 202 - 459 sacs per genotype ) . Different letters indicate significant differences in proportion of abnormal sacs in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . WT = wildtype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DIC images of abnormal embryo sacs in cycb1 mutant combinations. Red arrowheads indicate the visible nuclei in Col-0 embryo sacs (central and egg cells) and the corresponding structures in the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant. Scale bars: 20 μm. B. Quantification of the different abnormal embryo sac structures in cycb1 mutant combinations (n = 202-459 embryo sacs per genotype). Different letters indicate significant differences in the proportion of abnormal embryo sacs in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. WT = wildtype.


C . DIC of sacs 3 DAP with wildtype pollen ( x ) . Red arrowheads indicate visible sac nuclei in crosses with quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant as a donor , while control Col - 0 x Col - 0 cross exhibits a developing . Scale bars : 20 μm . D . of seed abortion in different cycb1 mutant combinations . Graph represents average seed abortion rate per ± SD of three biological replicates , n = 463 - 579 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA , followed by a Dunnett ' s multiple comparisons ( * * * * P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DIC images of embryo sacs 3 DAP with wildtype pollen (female x male). Red arrowheads indicate the visible embryo sac nuclei in the crosses with the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant as a female donor, while the control Col-0 x Col-0 cross exhibits a developing embryo. Scale bars: 20 μm. D. Quantification of seed abortion in different cycb1 mutant combinations. Graph represents the average seed abortion rate per plant ± SD of three biological replicates, n = 463-579 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


A , B . DAPI of pollen in cycb1 mutants , including pollen configurations found in cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cycb1 ; 4 - / - mutants ( B ) . Scale bars : 5 μm . C . of DAPI - stained pollen configurations in different cycb1 mutant combinations , n = 420 - 616 pollen grains per genotype . Different letters indicate significant differences in proportion of abnormal pollen ( uni - bicellular ) in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DAPI staining of pollen in cycb1 mutants, including pollen configurations found in cycb1;1-/- cycb1;2+/- cycb1;3+/- cycb1;4-/- mutants (B). Scale bars: 5 μm. C. Quantification of DAPI-stained pollen configurations in different cycb1 mutant combinations, n = 420-616 pollen grains per genotype. Different letters indicate significant differences in the proportion of abnormal pollen (uni- and bicellular) in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


D . Alexander of mature pollen indicating pollen viability . Scale bars : 5 μm . E . of Alexander - stained pollen viability , n = 403 - 498 pollen grains per genotype . Different letters indicate significant differences in proportion of dead pollen in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Alexander staining of mature pollen indicating pollen viability. Scale bars: 5 μm. E. Quantification of Alexander-stained pollen viability, n = 403-498 pollen grains per genotype. Different letters indicate significant differences in the proportion of dead pollen in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


A , B , C . Co - immunolocalization against tubulin ( magenta ) KNOLLE ( green ) in root meristematic . Nuclei were counterstained with DAPI for ( cyan ) . arrowheads indicate laggards in metaphase stage . Scale bars : 5 μm .

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A, B, C. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). White arrowheads indicate laggards in the metaphase stage. Scale bars: 5 μm.


D . of wildtype ( WT ) , double misplaced PPBs . Different letters indicate significant differences in proportions of different arrays per category in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . E . of PPBs with prominent perinuclear microtubules ( MTs ) . Boxes whiskers represent min to max values with median indicated as a central horizontal , n = 10 roots per genotype . Asterisks show significant differences in of PPBs with prominent perinuclear microtubules per root in an ANOVA followed by Dunnett ' s multiple comparisons ( * * P < 0 . 01 * * * * P < 0 . 0001 ) . F . of spindles with lagging chromosomes . Boxes whiskers represent min to max values with median indicated as a central horizontal , n = 10 roots per genotype . Asterisks show significant differences in of spindles with lagging chromosomes per root in a Kruskal - Wallis followed by Dunn ' s multiple comparisons ( * * P < 0 . 01 ns = non - significant ) .

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

D. Quantification of wildtype (WT), double and misplaced PPBs. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. E. Quantification of PPBs with prominent perinuclear microtubules (MTs). Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett's multiple comparisons test (** P < 0.01 and **** P < 0.0001). F. Quantification of spindles with lagging chromosomes. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (** P < 0.01 and ns = non-significant).


A . Co - immunolocalization against tubulin ( magenta ) KNOLLE ( green ) in root meristematic . Nuclei were counterstained with DAPI for ( cyan ) . Scale bars : 5 μm . B . of wildtype ( WT ) abnormal phragmoplasts . Different letters indicate significant differences in proportions of different arrays per category in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . C . of different mitotic stages in roots of different genotypes . Different letters indicate significant differences in proportions of different arrays per category in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). Scale bars: 5 μm. B. Quantification of wildtype (WT) and abnormal phragmoplasts. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. C. Quantification of the different mitotic stages in roots of the different genotypes. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.


A . Kinase assays against Histone H1 . Top middle panels indicate shorter longer exposures respectively of same kinase assays . Bottom panel is a CBB of Histone H1 showing equal loading of . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . B . Kinase assays against GIP1 . Top middle panels indicate shorter longer exposures respectively of same kinase assays . Bottom panel is a CBB of GIP1 showing equal loading of . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . C . Western blot against StrepIII - tagged to show loaded amounts of CDKs . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . Double CDKB2 ; 2 bands are likely due to a truncation of expressed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kinase assays against Histone H1. Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. B. Kinase assays against GIP1. Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of GIP1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. C. Western blot against StrepIII-tagged proteins to show loaded amounts of the CDKs. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. Double CDKB2;2 bands are likely due to a truncation of the expressed protein.


D . Time - lapse of confocal microscope pictures of root meristematic tagged with GFP - GIP1 in Col - 0 ( top panel ) cycb1 ; 1 cycb1 ; 2 ( bottom panel ) . GIP1 localizes at polar caps , followed by co - localization with microtubules at spindle phragmoplast arrays . In cycb1 ; 1 cycb1 ; 2 double mutants , GIP1 exhibited an abnormal localization , being found at spindle ( magenta arrowheads ) phragmoplast ( arrowheads ) midzones , which are normally devoid of . Scale bars : 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time-lapse of confocal microscope pictures of root meristematic cells tagged with GFP-GIP1 in Col-0 (top panel) and cycb1;1 cycb1;2 (bottom panel). GIP1 localizes at the nuclear polar caps, followed by co-localization with microtubules at the spindle and phragmoplast arrays. In cycb1;1 cycb1;2 double mutants, GIP1 exhibited an abnormal localization, being found at the spindle (magenta arrowheads) and phragmoplast (white arrowheads) midzones, which are normally devoid of the protein. Scale bars: 5 μm.


A - E Chemokine - galectin interactions were detected by using a solid - phase immunoassay . For this , 46 human CC CXC chemokines were adsorbed on nitrocellulose membranes stripes incubated in parallel with ( A ) TBS or ( B ) TBS containing biotinylated galectins ( representative image shows a processed tested with labeled Gal - 3 ) . Signals had been generated by using SA - HRP chemiluminescence reagents . ( C - D ) blots were subjected to densitometric , ( E ) independent experiments were combined ( binding chemokines in blue , Gal - 3 : n = 5 , Gal - 3 CRD : n = 5 , Gal - 1 n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Chemokine-galectin interactions were detected by using a solid-phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal-3). Signals had been generated by using SA-HRP and chemiluminescence reagents. (C-D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal-3: n = 5, Gal-3 CRD: n = 5, Gal-1 n = 4).


For SPR - experiments , ( F ) Gal - 3 ( density 650 RU ) , were immobilized increasing concentrations of CXCL12 were passed over flow . red curve represents a single - site fit to data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

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

For SPR-based experiments, (F) Gal-3 (density 650 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


For SPR - experiments , ( G ) Gal - 3 CRD ( density 1180 RU ) , were immobilized increasing concentrations of CXCL12 were passed over flow . red curve represents a single - site fit to data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

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

For SPR-based experiments, (G) Gal-3 CRD (density 1180 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


For SPR - experiments , ( H ) Gal - 1 ( density 130 RU ) were immobilized increasing concentrations of CXCL12 were passed over flow . red curve represents a single - site fit to data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

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

For SPR-based experiments, (H) Gal-1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


a - b Expansions of 1H - 15N HSQC spectra are overlaid Δδ values are plotted vs . acid sequences , along with secondary structures : ( a ) 10 µM 15N - enriched CXCL12 alone ( black peaks ) in presence of 330 µM Gal - 3 CRD ( red peaks ; assignments reported by Murphy et al . [ 74 ] ) ( b ) 30 µM 15N - enriched Gal - 3 CRD alone ( black peaks ) in presence of 500 µM CXCL12 ( red peaks ; assignments for Gal -

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a-b Expansions of 1H-15N HSQC spectra are overlaid and Δδ values are plotted vs. the amino acid sequences, along with secondary structures: (a) 10 µM 15N-enriched CXCL12 alone (black peaks) and in the presence of 330 µM Gal-3 CRD (red peaks; assignments reported by Murphy et al.[74]) and (b) 30 µM 15N-enriched Gal-3 CRD alone (black peaks) and in the presence of 500 µM CXCL12 (red peaks; assignments for Gal-3


a - c Jurkat T migrated in presence of 10 nM CXCL12 with increasing concentrations of ( a ) Gal - 3 , ( b ) Gal - 3 CRD , ( c ) Gal - 1 ( A - C : n = 4 ) .

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a-c Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of (a) Gal-3, (b) Gal-3 CRD, and (c) Gal-1 (A-C: n = 4).


D Human CD4 + T migrated in presence of 10 nM CXCL12 alone with 10 nM Gal - 3 ( green ) , Gal - 3 CRD ( green ) , Gal - 1 ( blue , n = 3 ) .

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

D Human CD4+ T cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3 (dark green), Gal-3 CRD (light green), and Gal-1 (blue, n = 3).


E - F ( E ) Jurkat T migrated to increasing concentrations of CXCL12 alone ( black ) CXCL12 in presence of 0 . 1 nM of Gal - 3 CRD ( green ) . ( F ) inhibitory effect of Gal - 3 CRD is shown as of chemotactic effect of CXCL12 ( E - F : n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (E) Jurkat T cells migrated to increasing concentrations of CXCL12 alone (black) and CXCL12 in the presence of 0.1 nM of Gal-3 CRD (light green). (F) The inhibitory effect of Gal-3 CRD is shown as percentage of the chemotactic effect of CXCL12 (E-F: n = 3).


G Jurkat T were allowed to migrate in presence of CXCL12 at increasing concentrations alone with 0 . 1 nM Gal - 1 or 100 nM Gal - 1 ( n = 3 ) .

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

G Jurkat T cells were allowed to migrate in the presence of CXCL12 at increasing concentrations alone and with 0.1 nM Gal-1 or 100 nM Gal-1 (n=3).


H Jurkat T did not migrate in sole presence of 1 nM to 1 µM Gal - 3 , Gal - 3 CRD ( both n = 5 ) , or Gal - 1 ( n = 4 ) .

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H Jurkat T cells did not migrate in the sole presence of 1 nM to 1 µM Gal-3, Gal-3 CRD (both n = 5), or Gal-1 (n = 4).


I viability of Jurkat T was assessed after incubation with 1 µM CXCL12 alone in presence of Gal - 3 , Gal - 3 CRD Gal - 1 ( n = 2 ) .

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

I The viability of Jurkat T cells was assessed after incubation with 1 µM CXCL12 alone and in the presence of Gal-3, Gal-3 CRD and Gal-1 (n = 2).


( J ) THP - 1 migrated in presence of 10 nM CXCL12 alone with 10 nM Gal - 3 , Gal - 3 CRD Gal -

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(J) THP-1 cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1


( K ) neutrophils migrated in presence of 10 nM CXCL12 alone with 10 nM Gal - 3 , Gal - 3 CRD Gal -

O 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) neutrophils migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1


L Human eosinophils migrated in presence of 10 nM CCL26 alone with 10 nM Gal - 3 , Gal - 3 CRD , Gal - 1 ( n = 3 ) .

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

L Human eosinophils migrated in the presence of 10 nM CCL26 alone and with 10 nM Gal-3, Gal-3 CRD, and Gal-1 (n = 3).


M CD4 + T migrated in presence of 1 nM CCL17 alone with equimolar concentrations of Gal - 3 , Gal - 3 CRD , Gal - 1 ( n = 4 ) .

M CD4+ T cells migrated in the presence of 1 nM CCL17 alone and with equimolar concentrations of Gal-3, Gal-3 CRD, and Gal-1 (n = 4).


A - B peritoneal recruitment of ( A ) CD45 + / CD115 - / Ly6G + neutrophils ( B ) CD45 + / CD115 + / Ly6Chi classical monocytes in C57BL / 6J mice was assessed 4 hours after intraperitoneal ( IP ) injection of 500 nM CXCL12 alone ( black ) in presence of 50 nM Gal - 3 ( green ; A - B : n = 7 mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The peritoneal recruitment of (A) CD45+/CD115-/Ly6G+ neutrophils and (B) CD45+/CD115+/Ly6Chi classical monocytes in C57BL/6J mice was assessed 4 hours after intraperitoneal (IP) injection of 500 nM CXCL12 alone (black) and in the presence of 50 nM Gal-3 (dark green; A-B: n = 7 mice).


C peritoneal recruitment of classical monocytes after IP injection of PBS ( n = 6 ) or TG in C57BL / 6J WT ( green , n = 10 mice ) Gal - 3 - / - ( black , n = 5 mice ) mice was assessed after 18 h . Where indicated , mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to experiment .

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

C The peritoneal recruitment of classical monocytes after IP injection of PBS (n = 6) or TG in C57BL/6J WT (dark green, n = 10 mice) and Gal-3-/- (black, n = 5 mice) mice was assessed after 18 h. Where indicated, the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment.


D of CXCL12 was determined by ELISA on peritoneal lavage normalized with from mesenterium ( n = 4 mice ) .

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

D The concentration of CXCL12 concentration was determined by ELISA on the peritoneal lavage normalized with levels from the mesenterium (n = 4 mice).


E CXCR4 expression on Ly6Chi monocytes of blood peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry indicated as mean ( MFI ) ( n = 4 mice ) .

E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity (MFI) (n = 4 mice).


A - B Jurkat T migrated in presence of 10 nM CXCL12 with increasing concentrations of E185A ( orange , n = 4 ) N222A ( red , n = 5 ) mutants of ( A ) Gal - 3 ( B ) Gal - 3 CRD .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of E185A (orange, n = 4) and N222A (red, n = 5) mutants of (A) Gal-3 and (B) Gal-3 CRD.


C - D Jurkat T migrated in presence of 10 nM CXCL12 with 10 nM ( C ) Gal - 3 ( D ) Gal - 3 CRD mutants as indicated ( C - D : n = 3 ) .

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

C-D Jurkat T cells migrated in the presence of 10 nM CXCL12 with 10 nM (C) Gal-3 and (D) Gal-3 CRD mutants as indicated (C-D: n = 3).


E Aggregation of Jurkat T in presence of 1 nM to 1 µM Gal - 3 Gal - 1 alone with 70 mM lactose , Gal - 3 CRD , was determined by flow cytometry ( n = 4 ) .

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

E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal-3 and Gal-1 alone and with 70 mM lactose, and Gal-3 CRD, was determined by flow cytometry (n = 4).


Jurkat T migrated with 10 nM CXCL12 alone in presence of ( F ) 1 µM Gal - 3 , ( G ) Gal - 1 , ( H ) Gal - 3 CRD in presence of 70 mM lactose or cellobiose or after treatment of with 150 µM DMJ .

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

Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (F) 1 µM Gal-3, (G) Gal-1, (H) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.


Jurkat T migrated with 10 nM CXCL12 alone in presence of ( I ) 0 . 1 nM Gal - 3 ( J ) Gal - 3 CRD in presence of 70 mM lactose or cellobiose or after treatment of with 150 µM DMJ .

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Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (I) 0.1 nM Gal-3 and (J) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.


a HEK transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone in presence of 10 nM Gal - 3 CRD followed by stimulation with forskolin , an activator of adenylate cyclase . Results are shown as luminescence relative to baseline ( RLU , example of n = 3 ) .

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a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD followed by stimulation with forskolin, an activator of adenylate cyclase. Results are shown as luminescence relative to baseline (RLU, example of n = 3).


b effect of 10 nM Gal - 3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation , was tested . Control experiments were performed as indicated ( n = 3 , four technical replicates ) .

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

b The effect of 10 nM Gal-3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation, was tested. Control experiments were performed as indicated (n = 3, four technical replicates).


c HEK transfected with a RlucII - conjugated CXCR4 an eYFP - β - arrestin 2 construct were stimulated with 10 nM CXCL12 alone in presence of 10 nM Gal - 3 CRD . Control experiments were performed as indicated . Results are given as net BRET ( i . e . of emissions at 535 / 485 nm minus of mock , n = 5 , three technical replicates ) .

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c HEK cells transfected with a RlucII-conjugated CXCR4 and an eYFP-β-arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD. Control experiments were performed as indicated. Results are given as the net BRET ratio (i.e. ratio of emissions at 535/485 nm minus the ratio of mock cells, n = 5, three technical replicates).


D Internalization of CXCR4 with 10 nM CXCL12 alone in presence of 10 nM Gal - 3 CRD was assessed by incubation with an APC - conjugated anti - CXCR4 antibody ( baseline signal = 100 % , n = 8 , two technical replicates ) .

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

D Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD was assessed by incubation with an APC-conjugated anti-CXCR4 antibody (baseline signal = 100%, n = 8, two technical replicates).


E 100 nM CXCL12 Gal - 3 were added to Jurkat T , their co - localization was assessed on surface by PLA . Control experiments were performed as indicated ( representative example of n = 3 ) . scale bar : 10 µm .

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E 100 nM CXCL12 and Gal-3 were added to Jurkat T cells, and their co-localization was assessed on the cell surface by PLA. Control experiments were performed as indicated (representative example of n = 3). White scale bar: 10 µm.


F - G Jurkat T were incubated with 100 nM of Gal - 3 CRD Alexa Fluor 555 alone , with 10 µM AMD 3100 , with 100 nM CXCL12 or with CXCL12 AMD 3100 . Experiments were performed ( f ) without ( g ) in presence of 70 mM lactose ( F - G : n = 3 , two technical replicates ) .

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F-G Jurkat T cells were incubated with 100 nM of Gal-3 CRD Alexa Fluor 555 alone, with 10 µM AMD 3100, with 100 nM CXCL12 or with CXCL12 and AMD 3100. Experiments were performed (f) without and (g) in the presence of 70 mM lactose (F-G: n = 3, two technical replicates).


H Jurkat T were incubated with 100 nM CXCL12 FITC alone with increasing concentrations of Gal - 3 CRD as indicated ( n = 4 , two technical replicates ) .

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H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal-3 CRD as indicated (n = 4, two technical replicates).


( a ) Kaplan - Meier survival curves for KRas ; Atg5 + / + ( n = 12 ) , KRas ; Atg5fl / + ( n = 19 ) KRas ; Atg5fl / fl ( n = 18 ) littermate mice injected intranasally with AdCre ( 2 . 5 × 107 p . f . u . ) . P0 . 01 ( log - rank ) between KRas ; Atg5fl / fl cohort

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The Kaplan-Meier survival curves for KRas;Atg5+/+ (n=12), KRas;Atg5fl/+ (n=19) and KRas;Atg5fl/fl (n=18) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). P0.01 (log-rank test) between the KRas;Atg5fl/fl cohort and controls


( b ) Western blot for Atg5 , LC3I LC3II p62 in purified lung tumour . Representative data are shown for tumour isolated from KRas ; Atg5fl / + KRas ; Atg5fl / fl mice 18 after AdCre inhalation . β - actin is shown as a loading control .

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(b) Western blot analysis for Atg5, LC3I and LC3II and p62 in purified primary lung tumour cells. Representative data are shown for tumour cells isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. β-actin is shown as a loading control.


( c ) Immunohistochemistry to detect LC3I , p62 Atg5 in tumours from KRas ; Atg5fl / + KRas ; Atg5fl / fl mice 18 after AdCre inhalation . Note highly elevated of LC3I p62 , both indicative of defective autophagy , in KRas ; Atg5fl / fl tumours . Scale bars , 50 μm . ( d , e ) Impaired formation of autophagosomes ( arrows ) in KRas ; Atg5fl / fl tumour .

(c) Immunohistochemistry to detect LC3I, p62 and Atg5 in tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. Note highly elevated levels of LC3I and p62, both indicative of defective autophagy, in KRas;Atg5fl/fl tumours. Scale bars, 50 μm. (d,e) Impaired formation of autophagosomes (arrows) in KRas;Atg5fl/fl tumour cells.


( d , e ) Impaired formation of autophagosomes ( arrows ) in KRas ; Atg5fl / fl tumour . ( d ) Representative electron microscopy are shown for KRas ; Atg5fl / + KRas ; Atg5fl / fl tumours18 after AdCre inhalation . Scale bars , 5 μm for upper 2 μm for lower panels . M , mitochondria ; * , lamellar bodies ( Corpuscula lamellariae ) , rare organelles containing surfactant lipoproteins characteristic for type II pneumocytes . ( e ) Mean percentages ( ± s . e . m . ) of tumour with detectable autophagosomes or autolysosomes , counted on electron microscopy . n ≥ 8 mice per genotype at least 20 intact for each electron microscopy section were counted . * * * P0 . 001 ( χ2 - of a generalized linear model with logit link assessing genotype effect ) .

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(d,e) Impaired formation of autophagosomes (arrows) in KRas;Atg5fl/fl tumour cells. (d) Representative electron microscopy images are shown for KRas;Atg5fl/+ and KRas;Atg5fl/fl tumours18 weeks after AdCre inhalation. Scale bars, 5 μm for upper and 2 μm for lower panels. M, mitochondria; *, lamellar bodies (Corpuscula lamellariae), rare cell organelles containing surfactant lipoproteins characteristic for type II pneumocytes. (e) Mean percentages (±s.e.m.) of tumour cells with detectable autophagosomes or autolysosomes, counted on electron microscopy images. n≥8 mice per genotype and at least 20 intact cells for each electron microscopy section were counted. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect).


( a ) Representative electron microscopy of autolysosomes ( left panels ) , autophagosomes ( middle panels ) mitophagy ( right panels ) , indicated by arrows , in lungtumour from control KRas ; Atg5fl / + mice18 after AdCre inhalation . Note near - to - complete absence of such structures in lungtumour from KRas ; Atg5fl / fl . Very rarely , we found with one autolysosome ( left panels ) or one single autophagosome ( middle panels ; arrows ) in KRas ; Atg5fl / fl tumours , whereas such structures can frequently be seen within single KRas ; Atg5fl / + tumour . Of note , presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig . 1e . Also note aberrant mitochondrial morphologies increased mitochondrial numbers in KRas ; Atg5fl / fl tumour . * indicates lamellar bodies . Scale bars , 2 μm . ( b ) Mean numbers ( ± s . e . m . ) of mitochondria per lungtumour from KRas ; Atg5fl / + KRas ; Atg5fl / fl mice18 after tumour induction . Numbers of mitochondria were determined by electron microscopy . n ≥ 8 mice per genotype a minimum of 20 intact were counted per sample . * * * P0 . 001 ( χ2 - assessing genotype effect in a generalized linear model with log link ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 electron microscopy images of autolysosomes (left panels), autophagosomes (middle panels) and mitophagy (right panels), all indicated by arrows, in lungtumour cells from control KRas;Atg5fl/+ mice18 weeks after AdCre inhalation. Note the near-to-complete absence of such structures in the lungtumour cells from KRas;Atg5fl/fl littermates. Very rarely, we found cells with one autolysosome (left panels) or one single autophagosome (middle panels; arrows) in KRas;Atg5fl/fl tumours, whereas such structures can frequently be seen within single KRas;Atg5fl/+ tumour cells. Of note, the presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig. 1e. Also note aberrant mitochondrial morphologies and increased mitochondrial numbers in KRas;Atg5fl/fl tumour cells. * indicates lamellar bodies. Scale bars, 2 μm. (b) Mean numbers (±s.e.m.) of mitochondria per lungtumour cell from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice18 weeks after tumour induction. Numbers of mitochondria were determined by electron microscopy. n≥8 mice per genotype and a minimum of 20 intact cells were counted per sample. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link).


( c ) Mitochondria ( arrows ) in KRas ; Atg5fl / + KRas ; Atg5fl / fl lung tumour 18 after AdCre inhalation . Note markedly altered mitochondrial , that is , disorganized matrix structures , rounding enlargement in autophagy - defective KRas ; Atg5fl / fl . Au , autophagosomes ; Au - L , autolysosome ; * , lamellar bodies . Scale bars , 2 μm .

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(c) Mitochondria (arrows) in KRas;Atg5fl/+ and KRas;Atg5fl/fl lung tumour cells 18 weeks after AdCre inhalation. Note markedly altered mitochondrial morphology, that is, disorganized matrix structures, rounding and enlargement in the autophagy-defective KRas;Atg5fl/fl cells. Au, autophagosomes; Au-L, autolysosome; *, lamellar bodies. Scale bars, 2 μm.


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

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


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

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


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

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


( c ) Tumour growth curves 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 ) .

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(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

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(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 ) .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 ) .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O 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) 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 - ) .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 B-CONTROLLED_VAR 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 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

(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 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

(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 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

(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 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 O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

(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 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 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 B-CONTROLLED_VAR 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 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 B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_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 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

(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 O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O 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 O O O O O O O O O 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 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

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

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


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

(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 O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O 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 B-MEASURED_VAR

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 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

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

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

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

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

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

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

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 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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 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

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 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 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 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

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.