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

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

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


( B ) Detection of NLRP3 expression ( top ) 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 .

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


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

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

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

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


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

O O O O O O O O O O O B-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 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 O

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O 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 ) analysis of rhNBD1 binding to P31 - 43 and 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 O O

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


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

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the intrinsic W401 fluorescence .

(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 the 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 O

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


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

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


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

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

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

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


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

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

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

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


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

O O 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

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


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

O O O 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 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 O O

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


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

O O 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 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 O O

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


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

O O 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 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

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


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

O O 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 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 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

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


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

O O 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 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

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


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

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 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

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


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

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

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


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

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 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 O O

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


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

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 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 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

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


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

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

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


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

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

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


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

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 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

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


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

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 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


( C ) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 ( ACT2 ) as a loading control .

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(C) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 (ACT2) as a loading control.


( D ) qPCR quantification of shGAG and flGAG - POL normalized to ACT2 and to GLYCERALDEHYDE - 3 - PHOSPHATE DEHYDROGENASE C SUBUNIT ( GAPC ) levels . qPCR was performed on n = 3 biological replicates ; bars : standard error . ( * * ) = p - value < 0 . 01 ( two - sided t - test between indicated values ) .

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(D) qPCR quantification of shGAG and flGAG-POL normalized to ACT2 and to GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPC) levels. qPCR was performed on n=3 biological replicates; bars: standard error. (**) = p-value < 0.01 (two-sided t-test between indicated values).


( E ) Western analysis of GAG and RDR6 with Coomassie ( coom . ) staining as a loading control . Arrow indicates cognate RDR6 protein band .

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(E) Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band.


( F - G ) sRNA - seq profiles from EVD de - repressed in the ddm1 ( F ) or ddm1 dcl1 ( G ) backgrounds . Different siRNA size categories are stacked . Nomenclature as in ( B ) .

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(F-G) sRNA-seq profiles from EVD de-repressed in the ddm1 (F) or ddm1 dcl1 (G) backgrounds. Different siRNA size categories are stacked. Nomenclature as in (B).


( B ) Expression levels of shGFP ( spliced ) and GFP - EVDint / ter - GUS ( unspliced ) transcripts , relative to ACT2 and AT4G26410 ( RHIP1 ) , in the WT or rdr6 background . qPCR was performed on three biological replicates and error bars represent the standard error on . ( * ) = p - value < 0 . 05 ( two - sided t - test against corresponding controls ) .

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(B) Expression levels of shGFP (spliced) and GFP-EVDint/ter-GUS (unspliced) transcripts, relative to ACT2 and AT4G26410 (RHIP1), in the WT or rdr6 background. qPCR was performed on three biological replicates and error bars represent the standard error on. (*) = p-value < 0.05 (two-sided t-test against corresponding controls).


( C ) sRNA - seq profile mapped on the genomic 35S : GFP - EVDint / ter - GUS locus . ( RPM ) Reads per million . Positions indicated in nucleotides ( nt ) from the start of the 35S sequence . Dashed vertical lines : shGFP and GFP - GUS 3 ' ends .

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(C) sRNA-seq profile mapped on the genomic 35S:GFP-EVDint/ter-GUS locus. (RPM) Reads per million. Positions indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGFP and GFP-GUS 3' ends.


( D ) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2 . qPCR was performed on three biological replicates and error bars represent the standard error . ( ns . ) = non - significant , ( * ) = p - value < 0 . 05 , ( * * ) = p - value < 0 . 01 , ( two - sided t - test between indicated samples / targets ) .

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(D) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2. qPCR was performed on three biological replicates and error bars represent the standard error. (ns.) = non-significant, (*) = p-value < 0.05, (**) = p-value < 0.01, (two-sided t-test between indicated samples/targets).


( E ) High and low molecular - weight RNA analysis of EVD GAG ( GAG - ex1 ) and EVD intron ( GAG - in ) in two independent T1 bulks from each indicated line . The filled arrows on the right hand - side or with an asterisk on the blots correspond to the transcripts depicted in ( A - C ) . ACT2 : loading control for mRNAs ; tasiR255 , miR173 and U6 : loading controls for sRNAs . Hybridizations for GAG - ex2 and POL probes are found in Supp . Fig . 5A .

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(E) High and low molecular-weight RNA analysis of EVD GAG (GAG-ex1) and EVD intron (GAG-in) in two independent T1 bulks from each indicated line. The filled arrows on the right hand-side or with an asterisk on the blots correspond to the transcripts depicted in (A-C). ACT2: loading control for mRNAs; tasiR255, miR173 and U6: loading controls for sRNAs. Hybridizations for GAG-ex2 and POL probes are found in Supp. Fig.5A.


( B ) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in ( A ) .

(B) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in (A).


( A ) Comparison of RNA isoforms and sRNA patterns generated by 35S : GFP - GUS and 35S : GFP - EVDint / ter - GUS . High and low molecular - weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background . mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig . 2A . EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs , respectively .

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(A) Comparison of RNA isoforms and sRNA patterns generated by 35S:GFP-GUS and 35S:GFP-EVDint/ter-GUS. High and low molecular-weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background. mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig.2A. EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs, respectively.


( B ) . Western blot analysis of the translation products from GFP and GFP - GUS transcripts . Coomassie ( coom . ) staining as a loading control . Black arrow : GFP - GUS fusion protein ; white arrow : GFP protein .

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(B). Western blot analysis of the translation products from GFP and GFP-GUS transcripts. Coomassie (coom.) staining as a loading control. Black arrow: GFP-GUS fusion protein; white arrow: GFP protein.


( C ) Nucleo - cytosolic distribution of 35S : EVD and 35S : GFP - EVDint / ter - GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR . RNA extracted from Total , nuclear ( Nucl ) and cytoplasmic ( Cyto ) fractions was reverse transcribed with random hexamers and oligo ( dT ) . snoRNA U5 is shown as a nuclear - only RNA control . ( D ) Same as in ( C ) but using exclusively oligo ( dT ) to reverse transcribe poly ( A ) + RNAs .

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

(C) Nucleo-cytosolic distribution of 35S:EVD and 35S:GFP-EVDint/ter-GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR. RNA extracted from Total, nuclear (Nucl) and cytoplasmic (Cyto) fractions was reverse transcribed with random hexamers and oligo(dT). snoRNA U5 is shown as a nuclear-only RNA control. (D) Same as in (C) but using exclusively oligo(dT) to reverse transcribe poly(A)+ RNAs.


( B ) RIBO - seq coverage profiles from 35S : EVD in WT or rdr6 . RPM : Reads per million . ( C ) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P - sites to calculate codon coverage . The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence .

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(B) RIBO-seq coverage profiles from 35S:EVD in WT or rdr6. RPM: Reads per million. (C) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P-sites to calculate codon coverage. The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence.


( B ) Schematic of marker - less GFP - tagging at the endogenous locus and representative live cell images of Mad1 - , Mad2 - , and Mad3 - GFP strains ( average intensity projections ) .

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(B) Schematic of marker-less GFP-tagging at the endogenous locus and representative live cell images of Mad1-, Mad2-, and Mad3-GFP strains (average intensity projections).


( C ) Representative images of single molecule mRNA FISH ( smFISH ) staining of S . pombe using probes against GFP ( red ) . DNA was stained with DAPI ( blue ) . The gamma - value was adjusted to make the cytoplasm visible ; cell shapes are outlined in blue . ( D ) Frequency distribution of mRNA numbers per cell determined by smFISH ; n = number of cells . Curves show fit to a Poisson distribution .

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(C) Representative images of single molecule mRNA FISH (smFISH) staining of S. pombe using probes against GFP (red). DNA was stained with DAPI (blue). The gamma-value was adjusted to make the cytoplasm visible; cell shapes are outlined in blue. (D) Frequency distribution of mRNA numbers per cell determined by smFISH; n = number of cells. Curves show fit to a Poisson distribution.


( E ) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP - tagged gene or the endogenous , untagged gene . Curves show fit to a Poisson distribution . The difference for mad1 + is statistically significant , that for mad2 + is not A lower mRNA number for untagged mad1 + was also observed in an independent strain .

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(E) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP-tagged gene or the endogenous, untagged gene. Curves show fit to a Poisson distribution. The difference for mad1+ is statistically significant, that for mad2+ is not A lower mRNA number for untagged mad1+ was also observed in an independent strain.


( F ) Co - staining by smFISH using probes against mad1 + and GFP either in a strain expressing mad1 + - GFP as a positive control or in a strain expressing wild - type mad1 + and mad2 + - GFP . Cytoplasmic mad1 + ( green ) or GFP mRNA spots ( magenta ) were quantified as co - localizing or not with the respective other . For the mad1 + - GFP strain , 544 cells and a total of 1 , 641 mad1 spots and 1 , 839 GFP spots were analyzed ; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm . For the mad1 + mad2 + - GFP strain , 571 cells and a total of 1 , 107 mad1 spots and 1 , 537 GFP spots were analyzed ; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm .

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(F) Co-staining by smFISH using probes against mad1+ and GFP either in a strain expressing mad1+-GFP as a positive control or in a strain expressing wild-type mad1+ and mad2+-GFP. Cytoplasmic mad1+ (green) or GFP mRNA spots (magenta) were quantified as co-localizing or not with the respective other. For the mad1+-GFP strain, 544 cells and a total of 1,641 mad1 spots and 1,839 GFP spots were analyzed; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm. For the mad1+ mad2+-GFP strain, 571 cells and a total of 1,107 mad1 spots and 1,537 GFP spots were analyzed; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm.


( A ) Cellular protein noise ( coefficient of variation , CV = std / mean ) in live - cell microscopy images of S . pombe ; n = 7 images ( Nmt1 - GFP ) , 11 ( Mad1 - GFP ) , 19 ( Mad2 - GFP ) , 10 ( Mad3 - GFP ) ; single images had 16 - 79 GFP - positive and 6 - 94 GFP - negative ( control ) cells . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Mad1 , Mad2 , and Mad3 all showed significantly lower noise than Nmt1 ( Wilcoxon rank sum test ; all p < 0 . 001 ) .

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(A) Cellular protein noise (coefficient of variation, CV = std / mean) in live-cell microscopy images of S. pombe; n = 7 images (Nmt1-GFP), 11 (Mad1-GFP), 19 (Mad2-GFP), 10 (Mad3-GFP); single images had 16-79 GFP-positive and 6-94 GFP-negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all p < 0.001).


( B ) Simulations of stochastic gene expression noise from selected mRNA / protein half - life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per cell , and a mean protein number of 6000 per cell . The x - axis of each graph shows time , the y - axis shows mRNA number per cell ( blue ) or protein number per cell ( black ) . ( C ) Theoretical prediction for the coefficient of variation ( CV = std / mean ) of the protein number per cell , assuming different mRNA and protein half - lives , using the same underlying model as in B . Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3 . 5 , and a mean protein number per cell of 6000 ( approx . 100 nM ) .

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(B) Simulations of stochastic gene expression noise from selected mRNA/protein half-life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6000 per cell. The x-axis of each graph shows time, the y-axis shows mRNA number per cell (blue) or protein number per cell (black). (C) Theoretical prediction for the coefficient of variation (CV = std / mean) of the protein number per cell, assuming different mRNA and protein half-lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6000 (approx. 100 nM).


( D ) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Lines are regression curves from generalized linear mixed model fits , excluding the measurements at t = 0 in order to accommodate for non - instantaneous labeling by 4tU . Act1 + and ecm33 + were used as long and short half - life controls , respectively ; qPCR was performed for the endogenous mRNAs . Half - lives ( 95 % confidence interval ) : mad1 + 5 . 6 min ( 4 . 3 - 8 . 4 ) , mad2 + 7 . 7 min ( 6 . 2 - 10 . 4 ) , mad3 + 5 . 2 min ( 4 . 3 - 6 . 9 ) , act1 + 61 . 8 min ( 37 . 2 - 172 . 3 ) , ecm33 + 5 . 0 min ( 4 . 5 - 5 . 7 ) .

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(D) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for non-instantaneous labeling by 4tU. Act1+ and ecm33+ were used as long and short half-life controls, respectively; qPCR was performed for the endogenous mRNAs. Half-lives (95 % confidence interval): mad1+ 5.6 min (4.3 - 8.4), mad2+ 7.7 min (6.2 - 10.4), mad3+ 5.2 min (4.3 - 6.9), act1+ 61.8 min (37.2 - 172.3), ecm33+ 5.0 min (4.5 - 5.7).


( E ) Protein abundances after translation shut - off with cycloheximide ( CHX ) ; n = 3 experiments , error bars = std . Lines indicate fit to a one - phase exponential decay . Cdc2 and Cdc13 were used as long and short half - life controls , respectively .

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

(E) Protein abundances after translation shut-off with cycloheximide (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one-phase exponential decay. Cdc2 and Cdc13 were used as long and short half-life controls, respectively.


( A ) The mean CSC value for each S . pombe gene ( CSCg ) relative to protein number per cell by mass spectrometry CSC was determined using the mRNA half - life data Colored dots highlight proteins of interest . For Mad2 and Bub1 , no protein abundance data was available . ( B ) Cumulative frequency distribution of the CSCg values for protein - coding S . pombe genes . The position of spindle assembly checkpoint genes is highlighted .

(A) The mean CSC value for each S. pombe gene (CSCg) relative to protein number per cell by mass spectrometry CSC was determined using the mRNA half-life data Colored dots highlight proteins of interest. For Mad2 and Bub1, no protein abundance data was available. (B) Cumulative frequency distribution of the CSCg values for protein-coding S. pombe genes. The position of spindle assembly checkpoint genes is highlighted.


( D ) Scatter plots of whole cell RNA counts versus cell length . Solid lines are regression curves from generalized linear mixed model fits ( gray : wild type , black : codon - optimized or ste13 ∆ ) . Dashed lines : 95 % bootstrap confidence bands for the regression curves . Model estimates of the ratio relative to wild - type mRNA are included with bootstrap 95 % confidence interval in brackets . Two to five replicates per genotype .

(D) Scatter plots of whole cell RNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets. Two to five replicates per genotype.


( E ) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( dark = ste13 + , light = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded area : 95 % bootstrap confidence band for ste13 + ; dashed lines : 95 % bootstrap confidence band for ste13 ∆ . Half - life estimates are included with 95 % bootstrap confidence intervals in brackets .

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

(E) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets.


( A ) Immunoblot of S . pombe protein extracts from cells expressing wild - type ( WT ) or codon - optimized ( co ) Mad2 - GFP or Mad3 - GFP probed with antibodies against GFP and Cdc2 ( loading control ) . Lanes 3 - 5 are a 1 : 1 dilution series of the extract from cells expressing the codon - optimized version . ( B ) Immunoblot of protein extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad2 , Mad3 , and tubulin ( loading control ) . A 1 : 1 dilution series was loaded for quantification . ( C ) Estimates of the protein concentration relative to wild - type conditions from experiments such as in ( A ) and ( B ) . Bars are experimental replicates , dots are technical replicates . Two - sided t - tests : p = 0 . 03 ( Mad2 - co ) , 0 . 004 ( Mad3 - co ) , 0 . 82 ( Mad2 ste13 ∆ ) , 0 . 15 ( Mad3 ste13 ∆ ) .

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

(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad2-GFP or Mad3-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a 1:1 dilution series of the extract from cells expressing the codon-optimized version. (B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad2, Mad3, and tubulin (loading control). A 1:1 dilution series was loaded for quantification. (C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Two-sided t-tests: p = 0.03 (Mad2-co), 0.004 (Mad3-co), 0.82 (Mad2 ste13∆), 0.15 (Mad3 ste13∆).


( D ) Whole - cell GFP concentration from individual live cell fluorescence microscopy experiments ( a . u . = arbitrary units ) . Boxes show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Codon - optimized concentration significantly higher than wild type for both genes ( GLMM ) . Mad2 - GFP : n = 468 and 413 ; Mad2 - co - GFP : n = 206 and 366 ; Mad3 - GFP : n = 224 and 127 ; Mad3 - co - GFP : n = 160 , 450 and 212 cells . ( E ) Representative images from one of the experiments in ( D ) . A single Z - slice is shown . Cells are outlined in gray .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxes show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly higher than wild type for both genes (GLMM). Mad2-GFP: n = 468 and 413; Mad2-co-GFP: n = 206 and 366; Mad3-GFP: n = 224 and 127; Mad3-co-GFP: n = 160, 450 and 212 cells. (E) Representative images from one of the experiments in (D). A single Z-slice is shown. Cells are outlined in gray.


( B ) Scatter plots of whole cell mRNA counts versus cell length . Solid lines are regression curves from generalized linear mixed model fits ( gray : wild type , black : codon - optimized or ste13 ∆ ) . Dashed lines : 95 % bootstrap confidence bands for the regression curves . Model estimates of the ratio relative to wild - type mRNA are included with bootstrap 95 % confidence interval in brackets . Two to three replicates 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

(B) Scatter plots of whole cell mRNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets. Two to three replicates per genotype.


( C ) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( dark = ste13 + , light = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded area : 95 % bootstrap confidence band for ste13 + ; dashed lines : 95 % bootstrap confidence band for ste13 ∆ . Half - life estimates are included with 95 % bootstrap confidence intervals in brackets .

(C) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets.


( D ) Comparison between mean CSC values for selected genes ( CSCg ) and mRNA half - life measured with or without deletion of ste13 + .

O O O O 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) Comparison between mean CSC values for selected genes (CSCg) and mRNA half-life measured with or without deletion of ste13+.


( A ) Immunoblot of S . pombe protein extracts from cells expressing wild - type ( WT ) or codon - optimized ( co ) Mad1 - GFP probed with antibodies against GFP and Cdc2 ( loading control ) . Lanes 3 - 5 are a dilution series of the extract from wild - type cells . ( B ) Immunoblot of protein extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad1 and tubulin ( loading control ) . A 1 : 1 dilution series was loaded for quantification . ( C ) Estimates of the protein concentration relative to wild - type conditions from experiments such as in ( A ) and ( B ) . Bars are experimental replicates , dots are technical replicates . Blue lines indicate the mean of all experiments . Two - sided t - tests : p = 0 . 005 ( Mad1 - co , n = 4 experimental replicates ) ; p = 0 . 16 ( Mad1 ste13 ∆ , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad1-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a dilution series of the extract from wild-type cells. (B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad1 and tubulin (loading control). A 1:1 dilution series was loaded for quantification. (C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Blue lines indicate the mean of all experiments. Two-sided t-tests: p = 0.005 (Mad1-co, n = 4 experimental replicates); p = 0.16 (Mad1 ste13∆, n = 2).


( D ) Whole - cell GFP concentration from individual live cell fluorescence microscopy experiments ( a . u . = arbitrary units ) . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Codon - optimized concentration significantly lower than wild type ( generalized linear mixed model ) . Mad1 - GFP : n = 197 and 224 ; Mad1 - co - GFP : n = 80 and 377 cells . ( E ) Representative images from one of the experiments in ( D ) . An average projection of three Z - slices is shown ; cells are outlined in gray .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly lower than wild type (generalized linear mixed model). Mad1-GFP: n = 197 and 224; Mad1-co-GFP: n = 80 and 377 cells. (E) Representative images from one of the experiments in (D). An average projection of three Z-slices is shown; cells are outlined in gray.


( F ) Live - cell imaging for time spent in mitosis . The alp7 + gene was deleted to increase the likelihood of spindle assembly checkpoint activation . Localization of Plo1 - tdTomato to spindle pole bodies was used to judge entry into and exit from mitosis Exp1 : n = 73 ( WT ) and 94 cells ( co ) ; Exp2 : n = 126 ( WT ) and 152 cells ( co ) . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Measurements for individual cells are shown in addition ( gray circles if measurement was exact , red triangles if end of mitosis was not captured because imaging ended ) . Difference between WT and co : p = 0 . 14 ( Exp1 ) and 0 . 15 ( Exp2 ) by Kolmogorov - Smirnov test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Live-cell imaging for time spent in mitosis. The alp7+ gene was deleted to increase the likelihood of spindle assembly checkpoint activation. Localization of Plo1-tdTomato to spindle pole bodies was used to judge entry into and exit from mitosis Exp1: n = 73 (WT) and 94 cells (co); Exp2: n = 126 (WT) and 152 cells (co). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Measurements for individual cells are shown in addition (gray circles if measurement was exact, red triangles if end of mitosis was not captured because imaging ended). Difference between WT and co: p = 0.14 (Exp1) and 0.15 (Exp2) by Kolmogorov-Smirnov test.


( A ) Top : Immunoprecipitation ( IP ) with anti - GFP or anti - Mad1 from extracts of haploid S . pombe cells expressing both untagged and GFP - tagged Mad1 , probed with antibodies against Mad1 and tubulin ; in = input ( 2 . 5 % of extract for IP ) , sup = supernatant after IP . Bottom : Comparison between the observed ( obs . ) and the expected ( expect . ) ratio between Mad1 - GFP and untagged Mad1 in the IP given their ratio in the input ; two and one experiment ( s ) , respectively . One more GFP - IP from the same strain was unquantifiable , because no second band was visible in the IP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Immunoprecipitation (IP) with anti-GFP or anti-Mad1 from extracts of haploid S. pombe cells expressing both untagged and GFP-tagged Mad1, probed with antibodies against Mad1 and tubulin; in = input (2.5 % of extract for IP), sup = supernatant after IP. Bottom: Comparison between the observed (obs.) and the expected (expect.) ratio between Mad1-GFP and untagged Mad1 in the IP given their ratio in the input ; two and one experiment(s), respectively. One more GFP-IP from the same strain was unquantifiable, because no second band was visible in the IP.


( C ) Top : Anti - GFP immunoprecipitation ( IP ) and Strep pull - down ( PD ) from extracts of diploid cells expressing Mad1 - GFP and Mad1 - Strep from the two endogenous loci ; membrane probed with anti - Mad1 ; in = input ( 7 % of extract for IP / PD ) , sup = supernatant after IP / PD . Bottom : as in ( A ) , 2 experiments each . The experiment shown at the top and two more GFP - IPs from the same strain were unquantifiable , because no second band was visible in the IP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Anti-GFP immunoprecipitation (IP) and Strep pull-down (PD) from extracts of diploid cells expressing Mad1-GFP and Mad1-Strep from the two endogenous loci; membrane probed with anti-Mad1; in = input (7 % of extract for IP/PD), sup = supernatant after IP/PD. Bottom: as in (A), 2 experiments each. The experiment shown at the top and two more GFP-IPs from the same strain were unquantifiable, because no second band was visible in the IP.


( D ) In vitro translation ( IVT ) of Mad1 - flag - His and untagged Mad1 in the presence of 35S - labelled Methionine and Cysteine , followed by Mad1 immunoprecipitation ( IP ) or His pull - down ( PD ) ; in = input ( 9 . 5 % of extract for IP / PD ) , sup = supernatant after IP / PD . An IVT with only untagged Mad1 was used to check for specificity of the His PD ( right side ) . Shown is the autoradiograph after SDS - PAGE with quantification of the Mad1 - flag - His to untagged Mad1 ratio in select lanes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro translation (IVT) of Mad1-flag-His and untagged Mad1 in the presence of 35S-labelled Methionine and Cysteine, followed by Mad1 immunoprecipitation (IP) or His pull-down (PD); in = input (9.5 % of extract for IP/PD), sup = supernatant after IP/PD. An IVT with only untagged Mad1 was used to check for specificity of the His PD (right side). Shown is the autoradiograph after SDS-PAGE with quantification of the Mad1-flag-His to untagged Mad1 ratio in select lanes.


( E ) Test for mRNA dimerization by single molecule mRNA FISH ; probes against mad1 + and GFP . Top : Schematic of genotypes . Bottom left : Intensity of cytoplasmic mad1 + mRNA spots in the different strains . For the 2 copy strain , a mad1 + spot was classified as mad1 + - GFP if it was colocalizing with a GFP spot , and as mad1 + otherwise . Colors as indicated in the schematic . Vertical solid line : peak of each density plot ; dashed line : theoretical position of a double - intensity peak . Number of spots analyzed : mad1 + ( 1 copy strain ) = 921 , mad1 + ( 2 copy strain ) = 637 , mad1 + - GFP ( 2 copy strain ) = 982 , mad1 + - GFP ( 1 copy strain ) = 1699 . Bottom right : Counts of cytoplasmic mad1 + or mad1 + - GFP mRNA from the same experiment with generalized linear mixed model fits as lines . Number of cells : 1 copy strain mad1 + = 478 , 2 copy strain = 327 , 1 copy strain mad1 + - GFP = 466 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Test for mRNA dimerization by single molecule mRNA FISH; probes against mad1+ and GFP. Top: Schematic of genotypes. Bottom left: Intensity of cytoplasmic mad1+ mRNA spots in the different strains. For the 2 copy strain, a mad1+ spot was classified as mad1+-GFP if it was colocalizing with a GFP spot, and as mad1+ otherwise. Colors as indicated in the schematic. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+ (1 copy strain) = 921, mad1+ (2 copy strain) = 637, mad1+-GFP (2 copy strain) = 982, mad1+-GFP (1 copy strain) = 1699. Bottom right: Counts of cytoplasmic mad1+ or mad1+-GFP mRNA from the same experiment with generalized linear mixed model fits as lines. Number of cells: 1 copy strain mad1+ = 478, 2 copy strain = 327, 1 copy strain mad1+-GFP = 466.


( F ) Experiment similar to ( E ) , except that cells expressing both mad1 + - GFP and mad3 + - GFP from the respective endogenous locus were probed with FISH probes against mad1 + and GFP mRNA . A GFP spot was classified as mad1 + - GFP if it was colocalizing with a mad1 + spot ( arrowheads ) , and as mad3 + - GFP otherwise . The intensity of GFP spots was quantified . Vertical solid line : peak of each density plot ; dashed line : theoretical position of a double - intensity peak . Number of spots analyzed : mad1 + - GFP = 987 , mad3 + - GFP = 1299 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Experiment similar to (E), except that cells expressing both mad1+-GFP and mad3+-GFP from the respective endogenous locus were probed with FISH probes against mad1+ and GFP mRNA. A GFP spot was classified as mad1+-GFP if it was colocalizing with a mad1+ spot (arrowheads), and as mad3+-GFP otherwise. The intensity of GFP spots was quantified. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+-GFP = 987, mad3+-GFP = 1299.


A Confocal images showing the basal regions of cochlear explants treated with either culture medium alone or medium containing 5 µM , 10 µM , or 20 µM CDDP for 3 days . The explants were labeled with Myosin 7A ( red ) to identify hair cells and NF200 ( green ) to highlight auditory nervefibers . Scale bar = 20 µm . nf : nerve fibers , OHCs : outer hair cells , IHCs : inner hair cells .

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

A Confocal images showing the basal regions of cochlear explants treated with either culture medium alone or medium containing 5 µM, 10 µM, or 20 µM CDDP for 3 days. The explants were labeled with Myosin 7A (red) to identify hair cells and NF200 (green) to highlight auditory nervefibers. Scale bar=20 µm. nf: nerve fibers, OHCs: outer hair cells, IHCs: inner hair cells.


B Dose - response curves of CDDP - induced loss of OHCs ( blue line ) and IHCs ( red line ) in basal cochlear regions . Data are expressed as mean ± SEM ( n = 5 cochleae per condition and per time point ) .

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

B Dose-response curves of CDDP-induced loss of OHCs (blue line) and IHCs (red line) in basal cochlear regions. Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).


C Effect over time on OHCs and IHCs treated with either culture medium alone ( light blue and red lines for OHCs and IHCs , respectively ) , or 10 µM CDDP ( blue and red lines for OHCs and IHCs , respectively ) . Data are expressed as mean ± SEM ( n = 5 cochleae per condition and per time point ) .

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

C Effect over time on OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively). Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).


D Representative Western blot analysis using antibodies against β - actin and cleaved caspase 3 in whole cochlear extracts . E Histogram representing the change in cleaved caspase 3 expression levels over time in control and CDDP group ( n = 6 cochleae per condition and per time point ) . Actin served as a loading control . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( * P ≤ 0 . 028 , * * P = 0 . 0007 , cochleae at the different times after CDDP exposure vs . control 12h ) .

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

D Representative Western blot analysis using antibodies against β-actin and cleaved caspase 3 in whole cochlear extracts.E Histogram representing the change in cleaved caspase 3 expression levels over time in control and CDDP group (n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P ≤ 0.028, **P=0.0007, cochleae at the different times after CDDP exposure vs. control 12h).