Figure 4Quantification of actin levels from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs ( n = 4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each sample ; * P = 0 . 021 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . Fractionation of G - and F - actin from CTRL and ECE2 KO COs and analysis via Westernblot additionally reveals an increase in G - at the expense F - actin upon ECE2 KO , suggesting a reduction in F - actin belt integrity ( n = 4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each ; * P = 0 . 021 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . The thickness of the apical F - actin belt is increased in ECE2 KO COs . C , Example images of apical F - actin belt in 60d old CTRL and ECE2 KO COs with F - actin labeled by AlexaFluor594 - conjugated Phalloidin ( Scalebar = 100 µm ) . D , Quantification of the thickness of apical F - actin belt in Fiji [ 86 ] by measuring the area of F - actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs ( Box plots : mean = red line , median = black line , box represents 25th and 75th percentiles , whiskers extend to 10th and 90th percentiles , all outliers are shown ; n = number of analysed ventricles in 2 batches ; Kruskal - Wallis One Way ANOVA on Ranks and Dunn ' s Pariwise Multiple Comparison : * * * P < 0 . 001 ) . IHC for ARL13B and F - ACTIN in 60d old CTRL and ECE2 KO COs shows disruption of honeycomb - like structure of the apical adherens belt and of apico - basal polarity in terms of apically localised primary cilium in ECE2 KO COs ( Scalebar = 10µm ) . The microtubule cytoskeleton is changed in COs upon ECE2 KO . Example images of ac - tub IHC in CTRL vs . ECE2 KO COs ( Scalebar = 100 µm ) . The microtubule cytoskeleton is changed in COs upon ECE2 KO . Quantification of ac - tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO ( n = number of analysed ventricles ; * P = 0 . 019 in One Way ANOVA and Tukey Pairwise Multiple Comparison ) . Apico - basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs . KO COs ( H ; Scalebar = 100 µm ) . Quantification of germinal zones for normally high ( " 1 " ) vs . reduced ( " 0 " ) apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 ( n = number of analysed ventricles from 2 batches ; * * * * P < 0 . 0001 in exact binomial test ) . Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43 - GFP reveals RG that delaminated ( white arrowheads ) and / or lost their bipolar morphology ( red arrowheads ) in the KO COs ( Scalebar = 25 µm ) .

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

Figure 4Quantification of actin levels from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each sample; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison).Fractionation of G- and F-actin from CTRL and ECE2 KO COs and analysis via Westernblot additionally reveals an increase in G- at the expense F-actin upon ECE2 KO, suggesting a reduction in F-actin belt integrity (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison).The thickness of the apical F-actin belt is increased in ECE2 KO COs. C, Example images of apical F-actin belt in 60d old CTRL and ECE2 KO COs with F-actin labeled by AlexaFluor594-conjugated Phalloidin (Scalebar=100 µm). D, Quantification of the thickness of apical F-actin belt in Fiji [86] by measuring the area of F-actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs (Box plots: mean=red line, median=black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of analysed ventricles in 2 batches; Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pariwise Multiple Comparison: ***P < 0.001).IHC for ARL13B and F-ACTIN in 60d old CTRL and ECE2 KO COs shows disruption of honeycomb-like structure of the apical adherens belt and of apico-basal polarity in terms of apically localised primary cilium in ECE2 KO COs (Scalebar = 10µm).The microtubule cytoskeleton is changed in COs upon ECE2 KO. Example images of ac-tub IHC in CTRL vs. ECE2 KO COs (Scalebar = 100 µm).The microtubule cytoskeleton is changed in COs upon ECE2 KO. Quantification of ac-tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO (n=number of analysed ventricles; *P = 0.019 in One Way ANOVA and Tukey Pairwise Multiple Comparison).Apico-basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs. KO COs (H; Scalebar = 100 µm). Quantification of germinal zones for normally high ("1") vs. reduced ("0") apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 (n=number of analysed ventricles from 2 batches; ****P < 0.0001 in exact binomial test).Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43-GFP reveals RG that delaminated (white arrowheads) and/or lost their bipolar morphology (red arrowheads) in the KO COs (Scalebar = 25 µm).


Figure 5Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6 + GFP + neural progenitors with significantly reduced localisation to bin2 ( A ) and increased localisation of Ctip2 + deep layer neurons to bin2 with reduction in bin5 ( B ) ( for binning strategy see Figure EV2B ; n = 3 CTRL and 5 ECE2 OX brains E13 - 14 ; data shown as bars with mean ± SEM ; * P < 0 . 05 and * * P < 0 . 01 in Student ' s t - test ) . Example images E13 - 14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions ( Scalebar = 100 µm ; electroporated cells shown in green ) . Ventricle marked by V . E13 - 16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2 + ( D ) progenitors and Ctip2 + deep - ( D ) and Satb2 + upper layer neurons ( E ) ( Scalebars = 100 µm ; electroporated cells shown in green ) . F , Quantification at 3dpe , counting the proportion of brains with normal vs . abnormal positioning of Sox2 + neural progenitors and Ctip2 + and Satb2 + neurons and that of brains with intact vs . disrupted basal membrane ( n = 5 CTRL and 8 ECE2 OX brains ; exact binomial test : * * * * P < 0 . 0001 ) . Ventricle marked by V . ECE2 OX in COs ( electroporated cells shown in green ; Scalebars = 50 µm ; n = number of CO ventricles analysed from 2 batches ) . G , Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3 + mitotic cells and NEUN + neuronal nuclei . H , Quantification of PH3 + cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background ( * P = 0 . 046 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . I , Quantification of GFP + neurons as proportion of all GFP + cells reveals an increase upon ECE2 OX in COs ( * P = 0 . 021 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . J , Quantification of ectopic neurons localising to the VZ ' relative to electroporated radial units ( GFP + cells in the VZ ' ) reveals a significant increase upon ECE2 OX ( * * * P < 0 . 001 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' . , Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning . K , Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe . L , Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs , thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX ( * * P = 0 . 002 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' .

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

Figure 5Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6+GFP+ neural progenitors with significantly reduced localisation to bin2 (A) and increased localisation of Ctip2+ deep layer neurons to bin2 with reduction in bin5 (B) (for binning strategy see Figure EV2B; n=3 CTRL and 5 ECE2 OX brains E13-14; data shown as bars with mean±SEM; *P < 0.05 and **P < 0.01 in Student's t-test).Example images E13-14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions (Scalebar = 100 µm; electroporated cells shown in green). Ventricle marked by V.E13-16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2+ (D) progenitors and Ctip2+ deep- (D) and Satb2+ upper layer neurons (E) (Scalebars = 100 µm; electroporated cells shown in green). F, Quantification at 3dpe, counting the proportion of brains with normal vs. abnormal positioning of Sox2+ neural progenitors and Ctip2+ and Satb2+ neurons and that of brains with intact vs. disrupted basal membrane (n=5 CTRL and 8 ECE2 OX brains; exact binomial test: ****P < 0.0001). Ventricle marked by V.ECE2 OX in COs (electroporated cells shown in green; Scalebars = 50 µm; n = number of CO ventricles analysed from 2 batches). G, Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3+ mitotic cells and NEUN+ neuronal nuclei. H, Quantification of PH3+ cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background (*P = 0.046 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). I, Quantification of GFP+ neurons as proportion of all GFP+ cells reveals an increase upon ECE2 OX in COs (*P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). J, Quantification of ectopic neurons localising to the VZ' relative to electroporated radial units (GFP+ cells in the VZ') reveals a significant increase upon ECE2 OX (***P < 0.001 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'., Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning. K, Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe. L, Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs, thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX (**P = 0.002 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'.


Figure 6Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO ( n = 3 batches of 60d old CTRL and ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating the cut - off ; names of example proteins are shown ) . Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins ( red ) in the secretome ( n = 3 batches of CTRL and ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating the cut - off ) . Non - significantly changed proteins of interest are highlighted in green below the cut - off curves .

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

Figure 6Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO (n=3 batches of 60d old CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off; names of example proteins are shown).Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins (red) in the secretome (n=3 batches of CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off). Non-significantly changed proteins of interest are highlighted in green below the cut-off curves.


Figure 1 ( A ) Immunoblots of mitochondrial fractions from siRNA ‐ transfected HEK293T cells ± CCCP . ( B ) Immunoblots against indicated proteases in siRNA ‐ transfected cells . ( C ) Quantification of full ‐ length PINK1 ( n = 4 ) and knockdown efficiency ( n = 2 ) compared with non ‐ targeting ( NT ) siRNA from experiments performed as in B . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA .

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

Figure 1(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP.(B) Immunoblots against indicated proteases in siRNA‐transfected cells.(C) Quantification of full‐length PINK1 (n=4) and knockdown efficiency (n=2) compared with non‐targeting (NT) siRNA from experiments performed as in B. *P0.05; **P0.01; ***P0.001. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


Figure 2 ( A ) Live confocal micrographs of siRNA ‐ transfected HEK293T cells ± CCCP stained with MitoTracker ( MTR ) Green and Deep Red ( DR ) ; Scale bar , 5 μm . ( B ) MTR DR / Green ratio from images as in A . N = 3 ; * P0 . 05 compared with NT siRNA . ( C ) Mitochondrial interconnectivity from images as in A , N = 3 . ( D ) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA ‐ transfected cells . Antimycin A with NT siRNA was used as a control . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; siRNA , short interfering RNA .

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

Figure 2(A) Live confocal micrographs of siRNA‐transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm.(B) MTR DR/Green ratio from images as in A. N=3; *P0.05 compared with NT siRNA.(C) Mitochondrial interconnectivity from images as in A, N=3.(D) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA‐transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; siRNA, short interfering RNA.


Figure 3 ( A ) Immunoblots of mitochondrial ( M ) and cytosolic ( C ) fractions from siRNA ‐ transfected HEK293T cells ± CCCP for 3 . 5 h , ± MG132 for the final hour . ( B ) Quantification of 52 ‐ kDa PINK1 from A ( with MG132 ) , n = 3 ; * P0 . 05 ; * * P0 . 001 compared with NT siRNA . ( C ) Mitochondria from siRNA ‐ transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate ( Resp . mix ) for 30 min at 37 ° C . al hour .

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

Figure 3(A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA‐transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour.(B) Quantification of 52‐kDa PINK1 from A (with MG132), n=3; *P0.05; **P0.001 compared with NT siRNA.(C) Mitochondria from siRNA‐transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C.al hour.


Figure 4 ( A ) Mitochondria from CCCP ‐ treated or siRNA ‐ transfected HEK293T cells were incubated with increasing concentrations of proteinase K ( PK ) . ( B ) Mitochondria from CCCP ‐ treated or MPPβ knockdown cells were incubated ± 0 . 1 M Na2CO3 before supernatant ( S ) and pellet ( P ) were separated by centrifugation . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA ; WB , western blot .

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

Figure 4(A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK).(B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.


Figure 5 ( A ) Live confocal micrographs of siRNA ‐ , GFP ‐ Parkin ‐ and OCT ‐ DsRed ‐ transfected HEK293T cells ± CCCP ; Scale bars , 20 μm ( low magnification ) and 5 μm ( high magnification ) . ( B ) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP ‐ Parkin distribution , n = 3 ; * P0 . 05 ; * * P0 . 01 compared with NT siRNA . ( C ) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA ‐ transfected cells ± CCCP . ( D ) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA ‐ transfected cells treated ± CCCP for 24 h , n = 3 . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; GFP , green fluorescent protein ; MPP , mitochondrial processing peptidase ; MTR , MitoTracker ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA .

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

Figure 5(A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification).(B) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP‐Parkin distribution, n=3; *P0.05; **P0.01 compared with NT siRNA.(C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP.(D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


Figure 1 ( A - B ) Expression patterns ( blue stain ) of GUS reporter genes driven by the SKM1 ( A ) or SKM2 ( B ) promoter in roots of 5 - day - old Col - 0seedlings . ( C ) Non - complementation of CLE45 insensitivity of the bam3mutant by BAM3expressed under control of the SKM1promoter ( 7 - day - old seedlings ; 2 independent transgenic lines per construct are shown ) . ( D ) Full CLE45 sensitivity of skm1or skm2 single and double mutant roots ( 7 - day - old seedlings ) . ( E ) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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

Figure 1(A-B) Expression patterns (blue stain) of GUS reporter genes driven by the SKM1(A) or SKM2(B) promoter in roots of 5-day-old Col-0seedlings.(C) Non-complementation of CLE45 insensitivity of the bam3mutant by BAM3expressed under control of the SKM1promoter (7-day-old seedlings; 2 independent transgenic lines per construct are shown).(D) Full CLE45 sensitivity of skm1or skm2 single and double mutant roots (7-day-old seedlings).(E) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


Figure 2 ( A ) Root phenotypes of 7 - day - old seedlings of indicated genotypes . ( B ) Primary root length of 7 - day - old seedlings . ( C ) Root meristem size in 5 - day - old seedlings . ( D ) Root meristem phenotype of 5 - day - old seedlings , confocal microscopy , propidium iodide - stained ( red ) . ( E ) Auxin activity as visualized by the constitutively expressed inverse DII - NLS - VENUS marker ( green ) in root meristems of 5 - day - old seedlings . ( F ) Gap cell frequency in brxsingle or brxmakr5 double mutants ( note : gap cells were absent from Col - 0 wild type or makr5single mutants ) . ( G ) Illustration of gap cells ( arrowheads ) in developing protophloem sieve element strands ( asterisks ) of brxsingle or brxmakr5 double mutants .

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

Figure 2(A) Root phenotypes of 7-day-old seedlings of indicated genotypes. (B) Primary root length of 7-day-old seedlings. (C) Root meristem size in 5-day-old seedlings.(D) Root meristem phenotype of 5-day-old seedlings, confocal microscopy, propidium iodide-stained (red).(E) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings.(F) Gap cell frequency in brxsingle or brxmakr5 double mutants (note: gap cells were absent from Col-0 wild type or makr5single mutants). (G) Illustration of gap cells (arrowheads) in developing protophloem sieve element strands (asterisks) of brxsingle or brxmakr5 double mutants.


Figure 3 ( A ) Primary root length of 7 - day - old seedlings of indicated genotypes in response to continuous CLE45 treatments . ( B ) Root meristem phenotypes of ( CLE45 - treated ) 5 - day - old seedlings ( asterisks : protophloem sieve element strands ) . Note absence of protophloem in CLE45 - treated Col - 0 wild type roots . ( C ) Transverse sections of ( CLE45 - treated ) 5 - day - old roots , just before the onset of protoxylem ( asterisks ) differentiation . Note failure of protophloem sieve elements ( arrowheads ) to differentiate in CLE45 - treated Col - 0 ( indicated by dense cytoplasm ) . ( D ) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5 - day - old Col - 0seedlings . ( E ) Corresponding transverse sections from different positions along the root meristem . ( F ) Expression of a MAKR5 - GFP fusion protein under control of the MAKR5 promoter in roots of 5 - day - old seedlings . ( G ) Corresponding optical sections illustrating MAKR5 - GFP expression in developing protophloem sieve elements ( asterisk ) and adjacent cell files . ( H ) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements ( CVP2 ) , but not by expression under control of a companion cell - specific promoter ( SUC2 ) ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) . ( I ) Subcellular localization of MAKR5 - GFP ( meristematic zone ) . Note plasma membrane association ( white , green arrowheads ) and dissociation from the cell wall ( red arrowhead ) upon osmosis .

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

Figure 3(A) Primary root length of 7-day-old seedlings of indicated genotypes in response to continuous CLE45 treatments.(B) Root meristem phenotypes of (CLE45-treated) 5-day-old seedlings (asterisks: protophloem sieve element strands). Note absence of protophloem in CLE45-treated Col-0 wild type roots.(C) Transverse sections of (CLE45-treated) 5-day-old roots, just before the onset of protoxylem (asterisks) differentiation. Note failure of protophloem sieve elements (arrowheads) to differentiate in CLE45-treated Col-0 (indicated by dense cytoplasm).(D) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings. (E) Corresponding transverse sections from different positions along the root meristem.(F) Expression of a MAKR5-GFP fusion protein under control of the MAKR5 promoter in roots of 5-day-old seedlings.(G) Corresponding optical sections illustrating MAKR5-GFP expression in developing protophloem sieve elements (asterisk) and adjacent cell files.(H) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements (CVP2), but not by expression under control of a companion cell-specific promoter (SUC2) (7-day-old seedlings; 3 independent transgenic lines per construct are shown).(I) Subcellular localization of MAKR5-GFP (meristematic zone). Note plasma membrane association (white, green arrowheads) and dissociation from the cell wall (red arrowhead) upon osmosis.


Figure 4 ( B ) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5 - day - old roots ( asterisks : protophloem sieve element strands ) . ( C ) Close up of protophloem sieve element strands expressing truncated MAKR5 - CITRINE fusion proteins under control of the UBQ10 promoter . ( D ) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant ( 7 - day - old seedlings ; 1 - 2 independent transgenic lines per construct are shown ) . ( E ) Sensitivity of makr5 mutant roots to continuous brassinolide ( BL ) treatment ( 7 - day - old seedlings ) . ( F ) Expression of MAKR5 - BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5 - day - old makr5 seedlings . ( G ) Complementation of CLE45 insensitivity of makr5 roots by BKI1 - MAKR5 , but not MAKR5 - BKI1 fusion protein ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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

Figure 4(B) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5-day-old roots (asterisks: protophloem sieve element strands).(C) Close up of protophloem sieve element strands expressing truncated MAKR5-CITRINE fusion proteins under control of the UBQ10 promoter.(D) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant (7-day-old seedlings; 1-2 independent transgenic lines per construct are shown).(E) Sensitivity of makr5 mutant roots to continuous brassinolide (BL) treatment (7-day-old seedlings).(F) Expression of MAKR5-BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5-day-old makr5 seedlings.(G) Complementation of CLE45 insensitivity of makr5 roots by BKI1-MAKR5, but not MAKR5-BKI1 fusion protein (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


Figure 5 ( A ) Increased expression of BAM3 - CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements ( asterisks ) of 5 - day - old bam3 roots upon CLE45 treatment . ( B ) Expression of BAM3 - CITRINE fusion protein under control of the BAM3 promoter in 5 - day - old Col - 0 roots or makr5 roots . ( C ) Expression of MAKR5 - GFP fusion protein under control of the MAKR5 promoter in 5 - day - old Col - 0 or bam3 roots . ( D ) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5 - day - old Col - 0seedlings upon CLE45 treatment . ( E ) Expression of MAKR5 - GFP fusion protein under control of the MAKR5 promoter in 5 - day - old roots upon CLE45 treatment ( 3 h ) . Note accumulation of MAKR5 - GFP in protophloem sieve elements ( magnified ) and its dependence on functional BAM3 and CLE45 . The frequency of representative observations per total observations is indicated . ( F ) Close up view of MAKR5 - GFP accumulation in the protophloem cell file upon CLE45 treatment . ( G - H ) Accumulation of MAKR5 - BKI1 fusion proteins in protophloem cells upon CLE45 treatment . The frequency of representative observations per total observations is indicated . ( I - J ) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter , showing mean total fluorescence ( I ) and plasma membrane to cytosol signal ratio ( J ) .

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Figure 5(A) Increased expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements (asterisks) of 5-day-old bam3 roots upon CLE45 treatment.(B) Expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots.(C) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old Col-0 or bam3 roots.(D) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings upon CLE45 treatment.(E) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old roots upon CLE45 treatment (3 h). Note accumulation of MAKR5-GFP in protophloem sieve elements (magnified) and its dependence on functional BAM3 and CLE45. The frequency of representative observations per total observations is indicated.(F) Close up view of MAKR5-GFP accumulation in the protophloem cell file upon CLE45 treatment.(G-H) Accumulation of MAKR5-BKI1 fusion proteins in protophloem cells upon CLE45 treatment. The frequency of representative observations per total observations is indicated. (I-J) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter, showing mean total fluorescence (I) and plasma membrane to cytosol signal ratio (J).


Figure 1A Dose - response binary food choice assay of saponin ( n ≥ 4 ) . B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla ( n ≥ 8 ) . C Sample traces of saponin - induced action potentials in ( B ) . D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla ( n ≥ 8 ) . E Sample traces from ( D ) . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis as a post hoc test to compare two sets of data ( * * P < 0 . 01 ) . The error bars represent SEMs .

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Figure 1A Dose-response binary food choice assay of saponin (n ≥ 4).B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla (n ≥ 8).C Sample traces of saponin-induced action potentials in (B).D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla (n ≥ 8). E Sample traces from (D). The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (**P < 0.01). The error bars represent SEMs.


Figure 2A Binary food choice assay with 26 Gr mutants for 5 % saponin ( n ≥ 4 ) . The error bars represent SEMs . B Concentration - dependent avoidance of saponin in control , Gr28bMi and Gr28b Df / Gr28bMi ( n ≥ 4 ) . C Amount of ingestion calculated at the indicated time points ( 15 , 30 , 60 , and 240 min ) using spectrophotogram analysis for control and 5 % saponin - containing food ( n = 8 ) . D , E PER assay for control and Gr28bMi flies in the ( D ) legs or ( E ) proboscis . Flies were initially given 2 % sucrose , then 2 % sucrose in combination with 5 % saponin ( n = 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis as a post hoc test to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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

Figure 2A Binary food choice assay with 26 Gr mutants for 5% saponin (n ≥ 4). The error bars represent SEMs.B Concentration-dependent avoidance of saponin in control, Gr28bMi and Gr28b Df/ Gr28bMi (n ≥ 4).C Amount of ingestion calculated at the indicated time points (15, 30, 60, and 240 min) using spectrophotogram analysis for control and 5% saponin-containing food (n = 8).D, E PER assay for control and Gr28bMi flies in the (D) legs or (E) proboscis. Flies were initially given 2% sucrose, then 2% sucrose in combination with 5% saponin (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (*P < 0.05, **P < 0.01).


Figure 3A Mapping analysis of all the sensilla following stimulation with 5 % saponin in control and Gr28bMi flies ( we followed Tanimura ' s nomenclature ) ( n ≥ 8 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) . B Sample traces of saponin - induced action potentials on S6 sensilla . C Tip recording analysis of S6 sensilla from control and Gr28bMi flies . Stimuli used are 0 . 2 % DEET , 1 . 0 mM coumarin , 0 . 5 mM quinine , 10 mM umbelliferone , 10 mM caffeine , 0 . 3 mM strychnine , 0 . 3 mM lobeline , 0 . 1 mM chloroquine , 0 . 2 mM denatonium , 0 . 5 mM papaverine , 0 . 1 mM berberine and 0 . 1 % SDS ( n ≥ 7 ) . The error bars represent SEMs . D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene ( hid ) , under Gr33a - GAL4 or Gr66a - GAL4 . All heterozygote controls ( UAS - hid / + , Gr33a - GAL4 / + , and Gr66a - GAL4 / + ) are shown ( n ≥ 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) . E Tip recording analysis of the indicated S - type sensilla and genotypes ( n ≥ 7 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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

Figure 3A Mapping analysis of all the sensilla following stimulation with 5% saponin in control and Gr28bMi flies (we followed Tanimura's nomenclature) (n ≥ 8). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).B Sample traces of saponin-induced action potentials on S6 sensilla.C Tip recording analysis of S6 sensilla from control and Gr28bMi flies. Stimuli used are 0.2% DEET, 1.0 mM coumarin, 0.5 mM quinine, 10 mM umbelliferone, 10 mM caffeine, 0.3 mM strychnine, 0.3 mM lobeline, 0.1 mM chloroquine, 0.2 mM denatonium, 0.5 mM papaverine, 0.1 mM berberine and 0.1% SDS (n ≥ 7). The error bars represent SEMs.D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr33a-GAL4 or Gr66a-GAL4. All heterozygote controls (UAS-hid/+, Gr33a-GAL4/+, and Gr66a-GAL4/+) are shown (n ≥ 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).E Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


Figure 4B RT - PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control . From left to right , the band of tubulin of wild - type , tubulin of Gr28bMi , DNA ladder , Gr28b of wild - type , and Gr28b of Gr28bMi . Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . The asterisks indicate significant differences from that of the Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s analysis ( * * P < 0 . 01 ) . C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4 / UAS systems . ( n ≥ 4 ) . D Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . The asterisks indicate significant differences from that of the Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s analysis ( * * P < 0 . 01 ) . D Saponin - induced action potential frequencies from the indicated transgenic flies on S6 sensilla ( n = 7 ) .

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

Figure 4B RT-PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control. From left to right, the band of tubulin of wild-type, tubulin of Gr28bMi, DNA ladder, Gr28b of wild-type, and Gr28b of Gr28bMi.Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4/UAS systems. (n ≥ 4).D Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). D Saponin-induced action potential frequencies from the indicated transgenic flies on S6 sensilla (n = 7).


Figure 5A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene ( hid ) , under Gr28b . c - GAL4 ( n = 4 ) . B Tip recording analysis of the indicated S - type sensilla and genotypes ( n ≥ 7 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * * P < 0 . 01 ) . C Expression of the Gr28b . c - GAL4 reporter in subsets of Gr66a - positive GRNs . First panel shows that all bitter responsive GRNs are labeled by Gr66a - I - GFP ( anti - GFP , green ) . Second panel shows the expression of the Gr28b . c - GAL4 , crossed with UAS - DsRed ( anti - DsRed , red ) . Third panel shows merged image of first and second panels . Forth panel shows a schematic diagram of sensilla arrangement . Red - marked sensilla indicate the expression of Gr28b . c - GAL4 reporter in second panel . Scale bars represent 40 μm .

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Figure 5A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr28b.c-GAL4 (n = 4).B Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (**P < 0.01). C Expression of the Gr28b.c-GAL4 reporter in subsets of Gr66a-positive GRNs. First panel shows that all bitter responsive GRNs are labeled by Gr66a-I-GFP (anti-GFP, green). Second panel shows the expression of the Gr28b.c-GAL4, crossed with UAS-DsRed (anti-DsRed, red). Third panel shows merged image of first and second panels. Forth panel shows a schematic diagram of sensilla arrangement. Red-marked sensilla indicate the expression of Gr28b.c-GAL4 reporter in second panel. Scale bars represent 40 μm.


Figure 6A , B Survival rate of ( A ) control and ( B ) Gr28bMi adult raised on control and the indicated concentrations of saponin - containing cornmeal diet ( n = 4 ) . C , D Survival rate based on binary food choice . control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM ( C ) or 200 mM fructose ( D ) mixed with 5 % or 7 . 5 % saponin . E , F Cumulative pupation rate of ( E ) control and ( F ) Gr28bMi larvae raised on the indicated saponin - containing food as well as control food ( n = 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from normal diet fed which were detected by a single - factor ANOVA with Scheffe ' s analysis ( * P < 0 . 05 , * * P < 0 . 01 ) .

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

Figure 6A, B Survival rate of (A) control and (B) Gr28bMi adult raised on control and the indicated concentrations of saponin-containing cornmeal diet (n = 4).C, D Survival rate based on binary food choice. control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM (C) or 200 mM fructose (D) mixed with 5% or 7.5% saponin.E, F Cumulative pupation rate of (E) control and (F) Gr28bMi larvae raised on the indicated saponin-containing food as well as control food (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from normal diet fed which were detected by a single-factor ANOVA with Scheffe's analysis (*P < 0.05, **P < 0.01).


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

Figure 1(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.(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).(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).(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).


Figure 2 ( 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 . ( 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 . 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 . ( I ) P31 - 43 induced modifications on NBD1 ATP binding site using the intrinsic W401 fluorescence . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.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.(I) P31-43 induced modifications on NBD1 ATP binding site using the intrinsic W401 fluorescence.(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.


Figure 3 ( 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 . 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 . 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 CFTRIncubation 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 antibodiesIncubation 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 . 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 . Incubation of Caco - 2 cells with P57 - 68 or P31 - 43 , in the presence or absence of pre - treatment with VX - 770 . ( L ) Immunoblot of phospho - PKA ( phPKA ) protein ; densitometric analysis of protein levels relative to β - actin . Mean ± SD of triplicates of independent experiments . * * p < 0 . 01 vs P31 - 43 , ° ° p < 0 . 001 vs VX - 770 ( ANOVA , Bonferroni post hoc test ) . Data information : The blots are representative of one experiment for group of treatment .

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

Figure 3(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.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.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 CFTRIncubation 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 antibodiesIncubation 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.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.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.


Figure 4Caco - 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 ) . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Caco-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).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.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.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.