( C ) Mean ± number ( upper panel ) ( lower panel ) of Iba - 1 positive in different brain regions from Npc1nmf164 wt mice ( n = 5 mice per group , > 30 per mouse , Student ´ s t - ) .

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(C) Mean ± SEM number (upper panel) and area (lower panel) of Iba-1 positive cells in different brain regions from Npc1nmf164 and wt mice (n = 5 mice per group, >30 cells per mouse, Student´s t-test).


( D ) Immunofluorescence against Iba - 1 in Cb of a NPC affected a control child . Scale bars , 50 μm . Graph shows mean ± of Iba - 1 positive ( n = 30 , Student ´ s t - ) .

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(D) Immunofluorescence staining against Iba-1 in the Cb of a NPC affected and a control child. Scale bars, 50 μm. Graph shows mean ± SEM area of Iba-1 positive cells (n = 30 cells, Student´s t-test).


( E ) Immunofluorescence against MBP in Cb of Npc1nmf164 wt mice . Scale bar , 50 μm . Graph shows mean ± number of MBP per ( n = 5 mice per group , Student ´ s t - ) .

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(E) Immunofluorescence staining against MBP in Cb of Npc1nmf164 and wt mice. Scale bar, 50 μm. Graph shows mean ± SEM number of MBP aggregates per area (n = 5 mice per group, Student´s t-test).


( F ) High magnification their 3D rendered replicates from cerebellar Npc1nmf164 microglia stained against Iba - 1 MBP containing myelin debris ( arrowheads ) . Scale bars , 10 μm ( up ) , 2 μm ( down ) . orthogonal projection of in right panels confirms presence of myelin debris inside underneath plasma . DAPI shows nuclei . Scale bars , 10 μm .

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(F) High magnification images and their 3D rendered replicates from cerebellar Npc1nmf164 microglia stained against Iba-1 and MBP containing myelin debris (arrowheads). Scale bars, 10 μm (up), 2 μm (down). The orthogonal projection of the cells in the right panels confirms the presence of myelin debris inside the cell and underneath the plasma membrane. DAPI staining shows cell nuclei. Scale bars, 10 μm.


( G ) Mean ± of microglia showing internalised myelin debris in wt Npc1nmf164 mice ( n = 5 mice per group ) .

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(G) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and Npc1nmf164 mice (n = 5 mice per group).


( H ) Immunofluorescence against F4 / 80 CathB in Cb of Npc1nmf164 mice . Scale bar , 30 μm . table shows mean ± of microglia presenting CathB in Npc1nmf164 wt mice . Below , magnified of selected areas . Scale bar , 20 μm .

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(H) Immunofluorescence staining against F4/80 and CathB in Cb of Npc1nmf164 mice. Scale bar, 30 μm. The table shows mean ± SEM percentage of microglia presenting CathB staining in Npc1nmf164 and wt mice. Below, magnified images of the selected areas. Scale bar, 20 μm.


( I ) Immunofluorescence against Iba - 1 CathB in Cb of a NPC . DAPI shows nuclei . Scale bar , 50 μm . A merged magnified image of selected is shown in right down panel , scale bar 10 μm . Table shows mean ± of microglia presenting CathB in NPC control children .

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(I) Immunofluorescence staining against Iba-1 and CathB in Cb of a NPC patient. DAPI shows cell nuclei. Scale bar, 50 μm. A merged magnified image of the selected area is shown in the right down panel, scale bar 10 μm. Table shows mean ± SEM percentage of microglia presenting CathB staining in NPC and control children.


Representative Western blot showing CARD14 in skin liver lysates of E18 . 5 pups . Actin is shown as a loading control .

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Representative Western blot showing CARD14 protein levels in skin and liver lysates of E18.5 pups. Actin is shown as a loading control.


Representative of K5creTg / + CARD14E138A + / + K5creTg / + CARD14E138ATg / + pups at E18 . 5 .

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Representative images of K5creTg/+ CARD14E138A+/+ and K5creTg/+ CARD14E138ATg/+ pups at E18.5.


Representative image of 6 month - old Malt1fl / fl K5cretg / + CARD14E138A + / + Malt1fl / fl K5cretg / + CARD14E138Atg / + mice .

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Representative image of 6 month-old Malt1fl/fl K5cretg/+ CARD14E138A+/+ and Malt1fl/fl K5cretg/+ CARD14E138Atg/+ mice.


ear thickness of untreated WT Malt1fl / fl K5creTg / + CARD14E138ATg / + mice ( between 4 7 months old ) . Each symbol represents one mouse ; represents mean value ( n ≥ 4 mice per group ) . WT = Malt1fl / fl K5cre + / + CARD14E138A + / + , Malt1fl / fl K5cre + / + CARD14E138ATg / + Malt1fl / fl K5creTg / + CARD14E138A + / + mice . Statistical difference between two groups was determined using a Mann - Whitney U - ( ns : p - value > 0 . 05 )

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Body weight and ear thickness of untreated WT and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ male and female mice (between 4 and 7 months old). Each symbol represents one mouse; the line represents the mean value (n≥4 mice per group). WT = Malt1fl/fl K5cre+/+ CARD14E138A+/+, Malt1fl/fl K5cre+/+ CARD14E138ATg/+ and Malt1fl/fl K5creTg/+ CARD14E138A+/+ mice. Statistical difference between two groups was determined using a Mann-Whitney U-test (ns: p-value>0.05)


Representative H & E - stained histological sections of ear skin tissue of Malt1fl / fl K5cre + / + CARD14E138A + / + Malt1fl / fl K5creTg / + CARD14E138ATg / + mice ( scale bar represents 100 μm ) .

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Representative H&E-stained histological sections of ear and skin tissue of Malt1fl/fl K5cre+/+ CARD14E138A+/+ and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ mice (scale bar represents 100 μm).


Changes in relative ear thickness upon CARD14E138A induction with tamoxifen . Malt1EKO = Malt1fl / fl . combined results of five independent experiments are shown ( Malt1 + / + WT n = 17 , Malt1fl / fl WT n = 8 , Malt1 + / + ieCARD14E138A n = 18 , Malt1fl / fl ieCARD14E138A n = 12 ) .

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Changes in relative body weight and ear thickness upon CARD14E138A induction with tamoxifen. Malt1EKO = Malt1fl/fl. The combined results of five independent experiments are shown (Malt1+/+ WT n=17, Malt1fl/fl WT n=8, Malt1+/+ ieCARD14E138A n=18, Malt1fl/fl ieCARD14E138A n=12).


Representative H & E - stained histological sections of ear tissue of tamoxifen - treated mice ( Scale bar represents 200 μm ) . Arrowheads indicate infiltrates of immune .

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Representative H&E-stained histological sections of ear tissue of tamoxifen-treated mice (Scale bar represents 200 μm). Arrowheads indicate infiltrates of immune cells.


Epidermal thickness measured on ear sections . Each symbol represents mean of at least ten measurements for each ear ; represents mean value . combined results of three independent experiments are shown .

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Epidermal thickness measured on ear sections. Each symbol represents the mean of at least ten measurements for each ear; the line represents the mean value. The combined results of three independent experiments are shown.


of infiltrating immune in ears of tamoxifen - treated mice using flow cytometry . of neutrophils ( CD45 + CD3 / CD19 - CD64 - CD11b + Ly6G + ) , eosinophils ( CD45 + CD3 / CD19 - CD64 - Ly6G - SiglecF + ) , T ( CD45 + CD3 / CD19 + , MHCII - ) , DCs ( CD45 + CD3 / CD19 - CD64 - MHCII + CD11c + ) in single suspensions of ear ( n ≥ 4 ) .

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Analysis of infiltrating immune cells in the ears of tamoxifen-treated mice using flow cytometry. Cell count of neutrophils (CD45+ CD3/CD19- CD64- CD11b+ Ly6G+), eosinophils (CD45+ CD3/CD19- CD64- Ly6G- SiglecF+), T cells (CD45+ CD3/CD19+, MHCII-), DCs (CD45+ CD3/CD19- CD64- MHCII+CD11c+) in single cell suspensions of the ear (n≥4).


mRNA expression of indicated genes in ears relative to reference genes ( Hprt1 , Rpl13a Tbp1 ) . Each symbol represents one mouse ; represents mean value ( n ≥ 3 ) .

mRNA expression levels of the indicated genes in ears relative to reference genes (Hprt1, Rpl13a and Tbp1). Each symbol represents one mouse; the line represents the mean value (n≥3).


Cleavage of MALT1 substrates CYLD BCL10 as analyzed by western blotting of lysates of ear tissue of mice four days after treatment with tamoxifen MALT1 inhibitor or vehicle . Actin is shown as a loading control . Each lane represents one mouse .

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Cleavage of MALT1 substrates CYLD and BCL10 as analyzed by western blotting of lysates of ear tissue of mice four days after treatment with tamoxifen and MALT1 inhibitor or vehicle. Actin is shown as a loading control. Each lane represents one mouse.


Changes in relative ear thickness in vehicle - MALT1 inhibitor - treated mice upon CARD14E138A induction . M1 inh = MALT1 inhibitor , WT = K14creERTg / + Rosa26 + / + , ieCARD14E138A = K14creERTg / + Rosa26LSL - CARD14 - E138A / + . Combined results of two independent experiments are shown ( WT + vehicle : n = 9 , WT + M1 inh : n = 8 , ieCARD14E138A + vehicle : n = 9 , ieCARD14E138A + M1 inh : n = 10 ) .

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Changes in relative ear thickness and body weight in vehicle- and MALT1 inhibitor-treated mice upon CARD14E138A induction. M1 inh= MALT1 inhibitor, WT = K14creERTg/+ Rosa26+/+, ieCARD14E138A = K14creERTg/+ Rosa26LSL-CARD14-E138A/+. Combined results of two independent experiments are shown (WT + vehicle: n=9, WT + M1 inh: n=8, ieCARD14E138A + vehicle: n=9, ieCARD14E138A + M1 inh: n=10).


Representative histological sections of ear tissue stained with hematoxylin and eosin


measurements of epidermal thickness of tamoxifen - treated mice . Combined results of two independent experiments are shown . Each symbol represents mean of at least ten epidermal thickness measurements for each ear ; represents mean value .

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measurements of epidermal thickness of tamoxifen-treated mice. Combined results of two independent experiments are shown. Each symbol represents the mean of at least ten epidermal thickness measurements for each ear; the line represents the mean value.


IL - 6 IL - 17a production by ear draining lymphocytes upon ex vivo restimulation with anti - CD3 / anti - CD28 for 3 days ( n ≥ 3 biological replicates ) .

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IL-6 and IL-17a production by ear draining lymphocytes upon ex vivo restimulation with anti-CD3/anti-CD28 for 3 days (n≥3 biological replicates).


showing relative mRNA expression of indicated genes in ear tissue normalized to reference genes . Values represent median of each group ( n ≥ 3 ) .

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Heatmap showing relative mRNA expression levels of the indicated genes in ear tissue normalized to reference genes. Values represent median of each group (n≥3).


Comparison of phenotype of ENCCs treated with intestinal extract from hypoganglionosis rats at 2 , 4 6 after BAC treatment . intestinal extracts from sham - treated guts were used as controls . ( A ) of ENCC - derived neurospheres on days 2 4 . ( B - C ) proliferation of ENCC - derived neurospheres is represented by neurosphere diameter ( B ) change in diameter on day 4 compared with day 2 ( C ) .

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Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (A) Morphology of the ENCC-derived neurospheres on days 2 and 4. (B-C) The proliferation of ENCC-derived neurospheres is represented by neurosphere diameter (B) and change in diameter on day 4 compared with day 2 (C).


Comparison of phenotype of ENCCs treated with intestinal extract from hypoganglionosis rats at 2 , 4 6 after BAC treatment . intestinal extracts from sham - treated guts were used as controls . ( D ) ENCC viability assessed by CCK - 8 .

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Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (D) ENCC viability assessed by CCK-8 assay.


Comparison of phenotype of ENCCs treated with intestinal extract from hypoganglionosis rats at 2 , 4 6 after BAC treatment . intestinal extracts from sham - treated guts were used as controls . ( E ) ENCCs number analyzed by with DAPI .

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Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (E) ENCCs number analyzed by nuclear staining with DAPI.


Comparison of phenotype of ENCCs treated with intestinal extract from hypoganglionosis rats at 2 , 4 6 after BAC treatment . intestinal extracts from sham - treated guts were used as controls . ( F ) Migration of ENCCs evaluated by transwell plate stain with crystal violet ,

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Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (F) Migration of ENCCs evaluated by transwell plate assay and stain with crystal violet,


Comparison of phenotype of ENCCs treated with intestinal extract from hypoganglionosis rats at 2 , 4 6 after BAC treatment . intestinal extracts from sham - treated guts were used as controls . ( G ) ENCC apoptosis determined by AnnexinV - FITC / PI stain flow cytometry .

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Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (G) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry.


Comparison of fecal microbial species inflammation in hypoganglionosis sham rats . For hypoganglionosis , fecal samples from narrow segments ( NS , BAC - treated , hypoganglionic ) expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( A - D ) Cluster at ( A ) phylum ( C ) genus . A column accumulation diagram of top 10 most abundant ( B ) phyla ( D ) genera .

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Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (A-D) Cluster analysis at the (A) phylum and (C) genus levels. A column accumulation diagram of the top 10 most abundant (B) phyla and (D) genera.


Comparison of fecal microbial species inflammation in hypoganglionosis sham rats . For hypoganglionosis , fecal samples from narrow segments ( NS , BAC - treated , hypoganglionic ) expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . Inter - group diversity to compare differences in ( H ) intestinal microbiota species indicated in red are linked to SCFAs production .

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Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. Inter-group diversity analysis to compare the differences in (H) intestinal microbiota The species indicated in red are linked to SCFAs production.


Comparison of fecal microbial species inflammation in hypoganglionosis sham rats . For hypoganglionosis , fecal samples from narrow segments ( NS , BAC - treated , hypoganglionic ) expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . metabolic mass spectrometry to compare differences ( I ) SCFAs metabolites between hypoganglionosis sham rats .

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Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. metabolic mass spectrometry to compare the differences (I) SCFAs metabolites between the hypoganglionosis and sham rats.


Comparison of fecal microbial species inflammation in hypoganglionosis sham rats . For hypoganglionosis , fecal samples from narrow segments ( NS , BAC - treated , hypoganglionic ) expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( J ) Representative HE to assess intestinal mucosal injury .

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Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (J) Representative HE staining and quantification to assess the intestinal mucosal injury.


Comparison of fecal microbial species inflammation in hypoganglionosis sham rats . For hypoganglionosis , fecal samples from narrow segments ( NS , BAC - treated , hypoganglionic ) expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( K ) Representative stain of CRP TNF - α .

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Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (K) Representative IHC stain and quantification of CRP and TNF-α levels.


Comparison of fecal microbial species inflammation in hypoganglionosis sham rats . For hypoganglionosis , fecal samples from narrow segments ( NS , BAC - treated , hypoganglionic ) expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( L ) Representative immunofluorescence stain of β - catenin . Nuclei were stained blue with DAPI .

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Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (L) Representative immunofluorescence stain and quantification of β-catenin levels. Nuclei were stained blue with DAPI.


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . diameter of ENCC - derived neurospheres on day 2 ( A ) .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. The diameter of ENCC-derived neurospheres on day 2 (A).


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . ENCC viability assessed by CCK - 8 ( B ) .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. ENCC viability assessed by CCK-8 assay (B).


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . ( C - D ) Migration of ENCCs using transwell plate stain with crystal violet ( C ) , scale bar , 200 µm . ( D ) of C . ( E - F ) ENCC apoptosis determined by AnnexinV - FITC / PI stain flow cytometry ( E ) . ( F ) of E .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (C-D) Migration of ENCCs using transwell plate assay and stain with crystal violet (C), scale bar, 200 µm. (D) Quantification of C. (E-F) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (E). (F) Quantification of E.


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . ( G ) Graphical protocol change in hypoganglionosis : untreated treated with ENCCs only , or ENCCs + FMT .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (G) Graphical protocol and weight change in the hypoganglionosis: untreated and treated with ENCCs only, or ENCCs+FMT.


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . ( H ) Laparoscopic indicating that same segments were treated with BAC in groups .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (H) Laparoscopic analysis indicating that the same segments were treated with BAC in all the groups.


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . ( I ) Delay in distal colonic transit time .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (I) Delay in distal colonic transit time.


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . Comparison of neuronal markers . ( J ) Immunofluorescence stain number of PGP9 . 5 + . Nuclei were stained blue with DAPI ; Triangles indicate PGP9 . 5 + .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. Comparison of neuronal markers. (J) Immunofluorescence stain and number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell.


Comparison of ENCC - derived neurospheres treated with intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) sham rat . Comparison of neuronal markers . ( K ) Representative immunofluorescence of Tuj1 GFP localization at injection adjacent sides . Asterisks triangles indicate Tuj1 + / GFP + Tuj1 + / GFP - , respectively . Nuclei were stained blue with DAPI . ( L - M ) Number of Tuj1 + , Tuj1 + / GFP + ( L ) Tuj1 + / GFP - ( M ) .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. Comparison of neuronal markers. (K) Representative immunofluorescence images of Tuj1 and GFP localization at the injection and adjacent sides. Asterisks and triangles indicate the Tuj1+/GFP+ cells and Tuj1+/GFP- cells, respectively. Nuclei were stained blue with DAPI. (L-M) Number of Tuj1+, Tuj1+/GFP+ cells (L) and Tuj1+/GFP- cells(M).


Comparison of fecal microbial species , short - chain fatty inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range ( , Antibiotic ) , broad - range followed by FMT ( FMT ) sham groups . ( A - D ) Cluster at ( A ) phylum ( C ) genus . Column accumulation diagrams of top abundant microbiota at ( B ) phylum ( D ) genus .

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Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (A-D) Cluster analysis at the (A) phylum and (C) genus. Column accumulation diagrams of the top abundant microbiota at the (B) phylum and (D) genus levels.


Comparison of fecal microbial species , short - chain fatty inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range ( , Antibiotic ) , broad - range followed by FMT ( FMT ) sham groups . ( G ) SCFAs subsets in feces intestinal extracts , analyzed by targeted mass spectrometry .

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Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (G) SCFAs subsets in the feces and intestinal extracts, analyzed by targeted mass spectrometry.


Comparison of fecal microbial species , short - chain fatty inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range ( , Antibiotic ) , broad - range followed by FMT ( FMT ) sham groups . ( H ) IL - 10 , IL - 17 , TNF - α in serum intestinal extracts , assessed by ELISA .

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Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (H) IL-10, IL-17, and TNF-α levels in serum and intestinal extracts, assessed by ELISA.


Comparison of fecal microbial species , short - chain fatty inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range ( , Antibiotic ) , broad - range followed by FMT ( FMT ) sham groups . Correlation of acetate , propionate or butyrate with IL - 17 in intestinal extracts .

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Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. Correlation analysis of acetate, propionate or butyrate with IL-17 in the intestinal extracts.


Phenotype of ENCCs treated with SCFAs . ( A ) ENCC viability assessed by CCK - 8 . Data are normalized to NC group .

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Phenotype analysis of ENCCs treated with SCFAs. (A) ENCC viability assessed by CCK-8 assay. Data are normalized to the NC group.


Phenotype of ENCCs treated with SCFAs . ( B - C ) Migration of ENCCs evaluated using transwell plate stain with crystal violet ( B ) ; Scale bars represent 200 µm . ( C ) Quantitative of B .

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Phenotype analysis of ENCCs treated with SCFAs. (B-C) Migration of ENCCs evaluated using transwell plate assay and stain with crystal violet (B); Scale bars represent 200 µm. (C) Quantitative analysis of B.


Phenotype of ENCCs treated with SCFAs . of different IL - 17 concentrations on ENCC viability ( D )

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Phenotype analysis of ENCCs treated with SCFAs. Analysis of different IL-17 concentrations on ENCC viability (D)


Phenotype of ENCCs treated with SCFAs . of different IL - 17 concentrations 50 ng / mL IL - 17 on ENCC migration ( E , F )

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Phenotype analysis of ENCCs treated with SCFAs. Analysis of different IL-17 concentrations 50 ng/mL IL-17 on ENCC migration (E, F)


Phenotype of ENCCs treated with SCFAs . ( G - H ) ENCC apoptosis determined by AnnexinV - FITC / PI stain flow cytometry ( G ) . ( H ) . of G .

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Phenotype analysis of ENCCs treated with SCFAs. (G-H) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (G). (H). Quantification of G.


( B ) Delay in distal colonic transit time .

(B) Delay in distal colonic transit time.


( C - D ) Representative immunofluorescent ( C ) ( D ) number of PGP9 . 5 + . Nuclei were stained blue with DAPI ; Triangles indicate PGP9 . 5 + .

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(C-D) Representative immunofluorescent images (C) and (D) number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell.


( E ) Representative immunofluorescent of Tuj1 GFP localization at injection adjacent sides . Asterisks triangles indicate Tuj1 + / GFP + Tuj1 + / GFP - , respectively .

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(E) Representative immunofluorescent images of Tuj1 and GFP localization at the injection and adjacent sides. Asterisks and triangles indicate the Tuj1+/GFP+ cells and Tuj1+/GFP- cells, respectively.


( F - G ) Number of Tuj1 + Tuj1 + / GFP + ( F ) Tuj1 + / GFP - ( G ) .

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(F-G) Number of Tuj1+ and Tuj1+/GFP+ cells (F) and Tuj1+/GFP- cells(G).


Volcano diagram shows differentially expressed genes ( DEGs ) in ENCCs after SCFAs IL - 17A intervention , with up - regulated down - regulated genes shown in yellow blue , respectively ( | log fold change | ≥ 0 . 5 adjusted p < 0 . 05 ) .

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Volcano diagram shows differentially expressed genes (DEGs) in ENCCs after SCFAs and IL-17A intervention, with up-regulated and down-regulated genes shown in yellow and blue, respectively (|log fold change|≥0.5 and adjusted p<0.05).


( E ) ENCC viability assessed by CCK - 8 after intervention with MEK1 / 2 inhibitor ( U0126 , 50 μM ) , p38 inhibitor ( SB203580 , 1 μM ) , JNK inhibitor ( SP600125 , 50 μM ) MER5 inhibitor ( BIX 02189 , 10 μM ) .

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(E) ENCC viability assessed by CCK-8 assay after intervention with MEK1/2 inhibitor (U0126, 50 μM), p38 inhibitor (SB203580, 1 μM), JNK inhibitor (SP600125, 50 μM) and MER5 inhibitor (BIX 02189, 10 μM).


Diameter analyses of ENCC - derived neurospheres ( F ) ,

Diameter analyses of the ENCC-derived neurospheres (F),


migration of ENCCs evaluated using transwell plate stain with crystal violet on day 2 ( G , H ) show that MEK1 / 2 inhibitor U0126 suppressed SCFA - induced ENCC proliferation migration .

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migration of ENCCs evaluated using transwell plate assay and stain with crystal violet on day 2 (G, H) show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration.


migration of ENCCs evaluated using transwell plate show that MEK1 / 2 inhibitor U0126 suppressed SCFA - induced ENCC proliferation migration .

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migration of ENCCs evaluated using transwell plate assay show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration.


B of INPP5Eprotein 72 h after transfection of N1E - 115 with control ( siControl ) or INPP5E - specific siRNAs ( siINPP5E # 1 or # 2 ) , as determined by immunoblot .

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B Levels of INPP5Eprotein 72 h after transfection of N1E-115 cells with control (siControl) or INPP5E-specific siRNAs (siINPP5E #1 or #2), as determined by immunoblot.


C N1E - 115 treated with siControl or siINPP5Es were cultured for 2 h in growth medium with or without 125 nM bafilomycin A1 ( Baf . A1 ) , then analyzed by immunoblot using anti - p62 , anti - LC3 , anti - GAPDH antibodies .

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C N1E-115 cells treated with siControl or siINPP5Es were cultured for 2 h in growth medium with or without 125 nM bafilomycin A1 (Baf.A1), and then analyzed by immunoblot using anti-p62, anti-LC3, and anti-GAPDH antibodies.


D Quantitation of signal intensities from immunoblots in C showing LC3 - II ( left ) or p62 ( right ) , following normalization to control proteinGAPDH . Because Baf . A1 inhibits autophagosome - lysosome fusion degradation within lysosomes via inhibition of vacuolar - type H + - ATPase , it is possible to measure autophagic flux , i . e . , degradation of autophagic substrates such as p62 LC3 - II , by calculating difference of signal intensities of these in presence absence of Baf . A1 . Results represent mean ± s . d . of three independent experiments . * * , P < 0 . 01 .

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D Quantitation of protein signal intensities from immunoblots in C showing LC3-II (left) or p62 (right) levels, following normalization to the control proteinGAPDH. Because Baf.A1 inhibits autophagosome-lysosome fusion and degradation within lysosomes via inhibition of vacuolar-type H+-ATPase, it is possible to measure autophagic flux, i.e., the degradation of autophagic substrates such as p62 and LC3-II, by calculating the difference of the signal intensities of these proteins in the presence and absence of Baf.A1. Results represent the mean ± s.d. of three independent experiments. **, P < 0.01.


E N1E - 115 treated with siControl or siINPP5Es were cultured in growth medium . were fixed stained with anti - LC3 antibodies analyzed by immunofluorescencemicroscopy . Bar , 10 µm .

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E N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LC3 antibodies and analyzed by immunofluorescencemicroscopy. Bar, 10 µm.


F Quantitation of number of LC3puncta per , as described in E ( mean ± s . d . ; n > 100 from three independent experiments ) . * * , P < 0 . 01 .

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F Quantitation of the number of LC3puncta per cell, as described in E (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.


G Electron micrographsin N1E - 115 treated with siControl or siINPP5Es were cultured in growth medium . Arrowheads indicate autophagosomes . Bar , 1 µm .

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G Electron micrographsin N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Arrowheads indicate autophagosomes. Bar, 1 µm.


A N1E - 115 treated with siControl , siINPP5E , or siAtg2a / 2b were cultured in growth medium . post - fraction ( PNS ) was separated into LSP , HSP , HSS fractions , then analyzed by immunoblots using anti - p62 , anti - LC3 , GAPDH antibodies . subfractions were treated with proteinase K ( Pro . K ) with or without Triton X - 100 . Quantitation of signal intensities from immunoblots showing LC3 - II ( left ) or p62 ( right ) , following normalization to control GAPDH . * * , P < 0 . 01 ; * , P < 0 . 05 .

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A N1E-115 cells treated with siControl, siINPP5E, or siAtg2a/2b were cultured in growth medium. The post-nuclear fraction (PNS) was separated into LSP, HSP, and HSS fractions, and then analyzed by immunoblots using anti-p62, anti-LC3, and GAPDH antibodies. The subfractions were treated with proteinase K (Pro. K) with or without Triton X-100. Quantitation of protein signal intensities from immunoblots showing LC3-II (left) or p62 (right) levels, following normalization to the control protein GAPDH. **, P < 0.01; *, P < 0.05.


B NIE - 115 stably expressing LAMP1 - mCherry treated with siControl or siINPP5Es were cultured for 2 h in growth medium containing 200 nM Torin1 with protease inhibitors ( 10 µg / ml Pepstatin A 10 µg / ml E - 64 - d ) . Because treatment of protease inhibitors inhibits lysosomal degradation , LC3puncta persist even ifautophagosomes fuse with lysosomes . were fixed stained with anti - LC3 antibodies , then analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . Bar , 10 µm . C Percentages of colocalization of LC3 dots with LAMP1 dots ( mean ± s . d . ; n > 20 from three independent experiments ) . * * , P < 0 . 01 .

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B NIE-115 cells stably expressing LAMP1-mCherry treated with siControl or siINPP5Es were cultured for 2 h in growth medium containing 200 nM Torin1 with protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d). Because the treatment of protease inhibitors inhibits lysosomal degradation, LC3puncta persist even ifautophagosomes fuse with lysosomes. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.C Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.


B N1E - 115 stably expressing mSt - INPP5E ( WT , D477N , ∆ CAAX , or 295 - 644 ) treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf . A1 for 2 h , then analyzed by immunoblot using anti - LC3 anti - GAPDH antibodies . mSt - INPP5Es used in this study were resistant to mouse siINPP5E because target of siRNA is different from human correspondent . C Quantitation of signal intensities from immunoblots in B , showing differences in LC3 - II in presence absence of Baf . A1 following normalization to control proteinGAPDH . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 ; * , P < 0 . 05 .

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B N1E-115 cells stably expressing mSt-INPP5E (WT, D477N, ∆CAAX, or 295-644) treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and then analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mSt-INPP5Es used in this study were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B, showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.


D NIE - 115 stably expressing mSt - INPP5E ( WT , D477N , or ΔCAAX ) were cultured in growth medium . were fixed stained with anti - LAMP1 antibodies , then analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . number showing to right side indicates colocalization rate of LAMP1 with mSt - INPP5E . Bar , 10 µm .

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D NIE-115 cells stably expressing mSt-INPP5E (WT, D477N, or ΔCAAX) were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. The number showing to the right side indicates the colocalization rate of LAMP1 with mSt-INPP5E. Bar, 10 µm.


A N1E - 115 treated with siControl or siINPP5Es were cultured in growth medium with 20 µg / ml DQ - BSA for 12 h chased for 2 h . were fixed stained with anti - LAMP1 antibodies analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . Bar , 10 µm . B Quantitation of signal intensities in A showing colocalization of DQ - BSA with LAMP1 ( means ± s . d . ; n > 50 from three independent experiments ) . Baf . A1 was used as a control for inhibition of delivery degradation of DQ - BSA via endocytic pathway . * * , P < 0 . 01 ; n . s . , non significant .

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A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg/ml DQ-BSA for 12 h and chased for 2 h. Cells were fixed and stained with anti-LAMP1 antibodies and analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.B Quantitation of signal intensities in A showing colocalization of DQ-BSA with LAMP1 (means ± s.d.; n > 50 cells from three independent experiments). Baf.A1 was used as a control for inhibition of delivery and degradation of DQ-BSA via the endocytic pathway. **, P < 0.01; n.s., non significant.


C N1E - 115 treated with siControl , siINPP5Es , or siCHMP5 were stimulated with 100 ng / ml EGF for indicated time periods , then analyzed by immunoblot using anti - EGFR anti - α - tubulin antibodies . of CHMP5 mRNA72 h after transfection of N1E - 115 with siControl or siCHMP5 , as analyzed by RT - PCR . D Quantitation of EGFR degradation in C . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 .

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C N1E-115 cells treated with siControl, siINPP5Es, or siCHMP5 were stimulated with 100 ng/ml EGF for the indicated time periods, and then analyzed by immunoblot using anti-EGFR and anti-α-tubulin antibodies. Levels of CHMP5 mRNA72 h after transfection of N1E-115 cells with siControl or siCHMP5, as analyzed by RT-PCR.D Quantitation of EGFR degradation ratio in C. Results represent means ± s.d. of three independent experiments. **, P < 0.01.


A NIE - 115 stably expressing mSt - 2xML1N or - 2xPLCδ1PH were cultured in growth medium with or without 200 nM Torin1 . were fixed stained with anti - LAMP1 or anti - LC3 antibodies , then analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . Bar , 10 µm .

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A NIE-115 cells stably expressing mSt-2xML1N or -2xPLCδ1PH were cultured in growth medium with or without 200 nM Torin1. Cells were fixed and stained with anti-LAMP1 or anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.


B NIE - 115 stably expressing mSt - 2xML1N treated with siControl or siINPP5Es were cultured in growth medium . were fixed stained with anti - LAMP1 antibodies , then analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . Bar , 10 µm .

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B NIE-115 cells stably expressing mSt-2xML1N treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.


C Quantitation of signal intensities in B showing mSt - 2xML1N colocalizing with LAMP1 ( means ± s . d . ; n > 100 from three independent experiments ) . * * , P < 0 . 01 ; * , P < 0 . 05 .

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C Quantitation of signal intensities in B showing mSt-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01; *, P < 0.05.


D N1E - 115 stably expressing GFP - 2xML1N treated with siControl or siINPP5E were transiently transfected with mSt - INPP5E ( WT , D477N ) . Quantitation of signal intensities showing GFP - 2xML1N colocalizing with LAMP1 ( means ± s . d . ; n > 20 from three independent experiments ) . * * , P < 0 . 01 ; * , P < 0 . 05 .

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D N1E-115 cells stably expressing GFP-2xML1N treated with siControl or siINPP5E were transiently transfected with mSt-INPP5E (WT, D477N). Quantitation of signal intensities showing GFP-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01; *, P < 0.05.


E NIE - 115 stably expressing mCherry - 2xFYVE treated with siControl or siINPP5Es were cultured in growth medium . were fixed stained with anti - LAMP1 antibodies , then analyzed by immunofluorescence microscopy . Insets show boxed areas at high magnification . Bar , 10 µm . F Quantitation of signal intensities in E showing mCherry - 2xFYVE colocalizing with LAMP1 ( means ± s . d . ; n > 100 from three independent experiments ) . * * , P < 0 . 01 .

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E NIE-115 cells stably expressing mCherry-2xFYVE treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.F Quantitation of signal intensities in E showing mCherry-2xFYVE colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.


A N1E - 115 treated with siControl or siINPP5Es were cultured in growth medium . were fixed stained with anti - LAMP1 antibodies Phalloidin , then analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . Bar , 10 µm . Quantitation of signal intensities showing Phalloidin colocalizing with LAMP1 ( means ± s . d . ; n > 40 from three independent experiments ) . * * , P < 0 . 01 .

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A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies and Phalloidin, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing Phalloidin colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.


B N1E - 115 treated with siControl or siINPP5Es were cultured in growth medium . were fixed stained with anti - phospho - Y421 cortactin anti - LAMP1 antibodies , then analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . Bar , 10 µm . Quantitation of signal intensities showing cortactin ( pY421 ) colocalizing with LAMP1 ( means ± s . d . ; n > 40 from three independent experiments ) . * * , P < 0 . 01 .

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B N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-phospho-Y421 cortactin and anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing cortactin (pY421) colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.


C N1E - 115 were treated with 0 . 5 µM Latrunculin A ( Lat A ) in growth medium for 6 h . were fixed stained with anti - LC3 antibodies , then analyzed by immunofluorescencemicroscopy . Bar , 10 µm . Quantitation of number of LC3puncta per . ( mean ± s . d . ; n > 100 from three independent experiments ) . * * , P < 0 . 01 .

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C N1E-115 cells were treated with 0.5 µM Latrunculin A (Lat A) in growth medium for 6 h. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Bar, 10 µm. Quantitation of the number of LC3puncta per cell. (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.


D NIE - 115 stably expressing LAMP1 - mCherry were treated with DMSO or 0 . 5 µM Lat A in growth medium for 4 h , subsequently cultured with 200 nM Torin1 protease inhibitors ( 10 µg / ml Pepstatin A 10 µg / ml E - 64 - d ) for 2 h ( Lat A treatment for total 6h ) . were fixed stained with anti - LC3 antibodies , then analyzed by immunofluorescencemicroscopy . Insets show boxed areas at high magnification . Bar , 10 µm . Percentages of colocalization of LC3 dots with LAMP1 dots ( mean ± s . d . ; n > 20 from three independent experiments ) . * * , P < 0 . 01 .

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D NIE-115 cells stably expressing LAMP1-mCherry were treated with DMSO or 0.5 µM Lat A in growth medium for 4 h, subsequently cultured with 200 nM Torin1 and protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d) for 2 h (Lat A treatment for total 6h). Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.


B N1E - 115 stably expressing indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf . A1 for 2 h , analyzed by immunoblot using anti - LC3 anti - GAPDH antibodies . mutants were resistant to mouse siINPP5E because target of siRNA is different from human correspondent . C Quantitation of signal intensities from immunoblots in B showing differences in LC3 - II in presence absence of Baf . A1 following normalization to control proteinGAPDH . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 ; * , P < 0 . 05 .

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B N1E-115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.


C38 IB3 - 1 transfected with GFP - LC3 plasmid in nutrient - rich or starvation medium . ( a ) Top : confocal microscopy of GFP . N , nucleus . Scale bar , 10 μm . Bottom : of containing more than five GFP - LC3 punctate dots per . Means ± s . d . ( n = 5 ) . Asterisk , P 0 . 001 versus C38 ; of variance ( ANOVA ) .

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C38 and IB3-1 cells transfected with GFP-LC3 plasmid in nutrient-rich or starvation medium. (a) Top: confocal microscopy of GFP. N, nucleus. Scale bar, 10 μm. Bottom: percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. (n = 5). Asterisk, P 0.001 versus C38 cells; analysis of variance (ANOVA).


( b ) Left : electron microscopy of starved C38 IB3 - 1 . right - are higher magnifications showing areas enriched in autophagosomes in C38 ( arrows ) perinuclear mitochondria in IB3 - 1 . N , nucleus . Scale bar , 250 nm . Right : numbers of autophagosomes per counted in 20 per experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus C38 ; ANOVA .

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(b) Left: electron microscopy of starved C38 and IB3-1 cells. The right-hand images are higher magnifications showing areas enriched in autophagosomes in C38 cells (arrows) and perinuclear mitochondria in IB3-1 cells. N, nucleus. Scale bar, 250 nm. Right: numbers of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus C38 cells; ANOVA.


( c ) - activated sorting ( FACS ) after with dye DiOC6 in starved C38 IB3 - 1 .

(c) Fluorescence-activated cell sorting (FACS) analysis after staining with fluorescent dye DiOC6 in starved C38 and IB3-1 cells.


( d , e ) Immunoblot of LC3 in starved C38 IB3 - 1 ( d ) IB3 - 1 ( e ) after incubation with E64d pepstatin A .

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(d, e) Immunoblot of LC3 in starved C38 and IB3-1 cells (d) and IB3-1 cells (e) after incubation with E64d and pepstatin A.


( f , g ) Immunoblots ( IB ) of polyubiquitin ( f ) in starved C38 IB3 - 1 . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(f, g) Immunoblots (IB) of polyubiquitin (f) in starved C38 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( f , g ) Immunoblots ( IB ) of p62 ( g ) in starved C38 IB3 - 1 . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

(f, g) Immunoblots (IB) of p62 (g) in starved C38 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( h ) Immunoblot of LC3 p62 in control ( n = 10 ) CF ( n = 10 ) human nasal mucosa . ( i ) Number of LC3 - positive dots ( per 104 mm − 2 of mucosa ) in control ( n = 10 ) CF ( n = 10 ) human nasal mucosa . Means ± s . d . Asterisk , P 0 . 01 versus controls ; ANOVA .

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(h) Immunoblot of LC3 and p62 in control (n = 10) and CF (n = 10) human nasal mucosa. (i) Number of LC3-positive dots (per 104 mm−2 of mucosa) in control (n = 10) and CF (n = 10) human nasal mucosa. Means ± s.d. Asterisk, P 0.01 versus controls; ANOVA.


( j ) Confocal microscopy of p62 in control CF human nasal mucosa . Representative from ten with CF . Nuclei counterstained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar , 10 μm .

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(j) Confocal microscopy of p62 in control and CF human nasal mucosa. Representative images from ten patients with CF. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 10 μm.


( k ) Number of LC3 - positive dots in lung tissues from wild - type ( WT ) CftrF508del mice . Means ± s . d . for lung tissues from ten mice per group . Asterisk , P 0 . 001 versus WT mice ; ANOVA . ( l ) Confocal microscopy of p62 in WT CftrF508del homozygous mice . Representative from at least five airway epithelial areas per mouse ( n = 10 mice per group ) . L , lumen . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm .

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(k) Number of LC3-positive dots in lung tissues from wild-type (WT) and CftrF508del mice. Means ± s.d. for lung tissues from ten mice per group. Asterisk, P 0.001 versus WT mice; ANOVA. (l) Confocal microscopy of p62 in WT and CftrF508del homozygous mice. Representative staining from at least five airway epithelial areas per mouse (n = 10 mice per group). L, lumen. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm.


( a - d ) Starved C38 IB3 - 1 . ( a - c ) Confocal microscopy of beclin 1 ( green ) hVps34 ( red ) ( a ) , calnexin ( green ) hVps34 ( red ) ( b ) , beclin 1 ( green ) HDAC6 ( red ) ( c , left ) or hVps34 ( green ) HDAC6 ( red ) ( c , right ) . N , nucleus . Scale bar , 10 μm . Yellow indicates co - localization .

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(a-d) Starved C38 and IB3-1 cells. (a-c) Confocal microscopy images of beclin 1 (green) and hVps34 (red) (a), calnexin (green) and hVps34 (red) (b), and beclin 1 (green) and HDAC6 (red) (c, left) or hVps34 (green) and HDAC6 (red) (c, right). N, nucleus. Scale bar, 10 μm. Yellow indicates co-localization.


( d ) Immunoblot of Ambra1 , hVps15 , hVps34 , Atg14L beclin 1 . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(d) Immunoblot of Ambra1, hVps15, hVps34, Atg14L and beclin 1. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( e ) Confocal microscopy of HA ( red ) calnexin ( green ) ( top ) or hVps34 ( red ) calnexin ( green ) ( bottom ) . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(e) Confocal microscopy images of HA (red) and calnexin (green) (top) or hVps34 (red) and calnexin (green) (bottom). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( f ) Left : confocal of LC3 - positive dots . N , nucleus . Scale bar , 10 μm . Right : of containing more than five LC3 - positive dots . At least 60 were counted in each experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus transfected with empty vector ; ANOVA .

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(f) Left: confocal images of LC3-positive dots. N, nucleus. Scale bar, 10 μm. Right: percentage of cells containing more than five LC3-positive dots. At least 60 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA.


( g ) Left : electron microscopy of HA - beclin 1 - transfected IB3 - 1 shows areas enriched in autophagosomes ( arrows ) . Scale bar , 250 nm . Right : number of autophagosomes per ; 20 were counted in each experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus transfected with empty vector ; ANOVA .

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(g) Left: electron microscopy of HA-beclin 1-transfected IB3-1 cells shows areas enriched in autophagosomes (arrows). Scale bar, 250 nm. Right: number of autophagosomes per cell; 20 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA.


( h ) Immunoblot of HA LC3 . ( i ) Immunoblot of p62 . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(h) Immunoblot of HA and LC3. (i) Immunoblot of p62. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( j ) Immunoblot of beclin 1 in WT CftrF508del mice . Representative of seven mice for each group .

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(j) Immunoblot of beclin 1 in WT and CftrF508del mice. Representative of seven mice for each group.


( k , l ) CftrF508del mice ( n = 3 per group ) were administered intranasally with a lentiviral vector encoding beclin 1 ( LV - beclin 1 ) or GFP ( LV - GFP ) . ( k ) LC3 - positive dots per mm2 of lung tissues . Means ± s . d . for lung tissues from three mice per group . ( l ) Confocal microscopy of p62 . L , lumen . Representative stainings from at least five airway epithelial areas per mouse . Scale bar , 10 μm .

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(k, l) CftrF508del mice (n = 3 per group) were administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (k) LC3-positive dots per mm2 of lung tissues. Means ± s.d. for lung tissues from three mice per group. (l) Confocal microscopy micrographs of p62. L, lumen. Representative stainings from at least five airway epithelial areas per mouse. Scale bar, 10 μm.


( a ) IB3 - 1 cultured with 250 μM cystamine or transfected with either 50 nM human TG2 siRNA or scrambled oligonucleotides . IP , immunoprecipitation ; IB , immunoblot .

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(a) IB3-1 cells cultured with 250 μM cystamine or transfected with either 50 nM human TG2 siRNA or scrambled oligonucleotides. IP, immunoprecipitation; IB, immunoblot.


( b ) IB3 - 1 cultured with proteasome inhibitor MG132 ( 50 μM ) .

(b) IB3-1 cells cultured with the proteasome inhibitor MG132 (50 μM).


( c , d ) IB3 - 1 transfected with either human TG2 siRNA or scrambled oligonucleotides followed by incubation with MG132 . ( c ) immunostained with anti - beclin 1 ( green ) anti - HDAC - 6 ( red ) antibodies . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(c, d) IB3-1 cells transfected with either human TG2 siRNA or scrambled oligonucleotides followed by incubation with MG132. (c) Cells immunostained with anti-beclin 1 (green) and anti-HDAC-6 (red) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( c , d ) IB3 - 1 transfected with either humanTG2 siRNA or scrambled oligonucleotides followed by incubation with MG132 . ( d ) FRET of beclin 1 - Alexa 546 ( Al546 ) after HDAC6 - Cy5 photobleaching . F / F0 indicates post - bleaching / pre - bleaching of Al546 / Cy5 .

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(c, d) IB3-1 cells transfected with either humanTG2 siRNA or scrambled oligonucleotides followed by incubation with MG132. (d) FRET analysis of beclin 1-Alexa 546 (Al546) fluorescence after HDAC6-Cy5 photobleaching. F/F0 indicates the post-bleaching/pre-bleaching fluorescence ratio of Al546/Cy5.


( e ) IB3 - 1 were transfected with either human TG2 siRNA or scrambled oligonucleotides . Immunoblot of TG2 , Ambra - 1 , hVps15 , hVps34 , Atg14L beclin 1 in IB3 - 1 grown in starvation . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(e) IB3-1 cells were transfected with either human TG2 siRNA or scrambled oligonucleotides. Immunoblot of TG2, Ambra-1, hVps15, hVps34, Atg14L and beclin 1 in IB3-1 cells grown in starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( f ) IB3 - 1 C38 were transfected with either 1 μg of pLPCX - WT - TG or pLPCX - Cys277Ser - TG or empty vector . Immunoblot of beclin 1 . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(f) IB3-1 and C38 cells were transfected with either 1 μg of pLPCX-WT-TG or pLPCX-Cys277Ser-TG or the empty vector. Immunoblot of beclin 1. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( g , h ) IB3 - 1 were treated with cystamine or 10 mM EUK - 134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides . ( g ) of containing more than five GFP - LC3 punctate dots . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus control ; ANOVA .

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(g, h) IB3-1 cells were treated with cystamine or 10 mM EUK-134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides. (g) Percentage of cells containing more than five GFP-LC3 punctate dots. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA.


( g , h ) IB3 - 1 were treated with cystamine or 10 mM EUK - 134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides . ( h ) Left : electron of IB3 - 1 showing areas enriched in autophagosomes ( arrows ) on PIASy gene silencing , EUK - 134 or cystamine . Scale bar , 100 nm . Right : number of autophagosomes per counted in 20 per experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus control ; ANOVA .

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(g, h) IB3-1 cells were treated with cystamine or 10 mM EUK-134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides. (h) Left: electron micrographs of IB3-1 cells showing areas enriched in autophagosomes (arrows) on PIASy gene silencing, EUK-134 or cystamine. Scale bar, 100 nm. Right: number of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA.


( i ) IB3 - 1 treated with cystamine . Immunoblot of p62 .

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(i) IB3-1 cells treated with cystamine. Immunoblot of p62.


( j ) IB3 - 1 treated with cystamine or EUK - 134 or transfected with human PIASy siRNA or scrambled oligonucleotides . Immunoblot of beclin 1 ( left ) PIASy ( right ) . Uncropped of blots are shown in Supplementary Information , Fig . S10 . experiments were repeated three times . β - actin was used as loading control for blots . Densitometric of blots quantitative measurement of co - localizations are shown in Supplementary Information , Fig . S9 .

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(j) IB3-1 cells treated with cystamine or EUK-134 or transfected with human PIASy siRNA or scrambled oligonucleotides. Immunoblot of beclin 1 (left) and PIASy (right). Uncropped images of blots are shown in Supplementary Information, Fig. S10. The experiments were repeated three times. β-actin was used as loading control for all blots. Densitometric analysis of blots and quantitative measurement of co-localizations are shown in Supplementary Information, Fig. S9.


( a , b ) C38 were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides ( a , b ) or cultured with CFTRinh - 172 ( b ) in presence or absence of cystamine ( 250 μM ) or EUK - 134 ( 10 mM ) . ( a ) Immunoblot of beclin 1 , LC3 p62 in C38 on starvation . Uncropped of blots are shown in Supplementary Information , Fig . S10 . ( b ) of containing more than five GFP - LC3 punctate dots per . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus scrambled oligonucleotides ; circle , P 0 . 01 versus CFTR siRNA or CFTRinh - 172 ; ANOVA .

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(a, b) C38 cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides (a, b) or cultured with CFTRinh-172 (b) in the presence or absence of cystamine (250 μM) or EUK-134 (10 mM). (a) Immunoblot of beclin 1, LC3 and p62 in C38 cells on starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (b) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus scrambled oligonucleotides; circle, P 0.01 versus CFTR siRNA or CFTRinh-172; ANOVA.


( c - g ) A549 were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( c ) Immunoblot of TG2 PIASy .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine. (c) Immunoblot of TG2 and PIASy.


( c - g ) A549 were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( d ) FRET of TG2 - Alexa 546 after SUMO - 1 Cy5 photobleaching .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(d) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching.


( c - g ) A549 were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( e ) Immunoblot of beclin 1 LC3 in A549 after starvation . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(e) Immunoblot of beclin 1 and LC3 in A549 cells after starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( c - g ) A549 were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( f ) of containing more than five GFP - LC3 punctate dots per . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 ; two asterisks , P 0 . 01 ; ANOVA . ( g ) Immunostaining with anti - p62 ( red ) anti - GFP ( green ) antibodies in starved A549 . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm . Each experiment was repeated three times .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(f) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001; two asterisks, P 0.01; ANOVA. (g) Immunostaining with anti-p62 (red) and anti-GFP (green) antibodies in starved A549 cells. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. Each experiment was repeated three times.


( a , b ) IB3 - 1 were transfected with either HA - tagged human beclin 1 , empty vector , p62 siRNA or scrambled oligonucleotides followed by rosiglitazone ( 10 μM ) . ( a ) were immunostained with anti - HDAC6 ( red ) anti - PPAR - γ ( green ) antibodies . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(a, b) IB3-1 cells were transfected with either HA-tagged human beclin 1, empty vector, p62 siRNA or scrambled oligonucleotides followed by rosiglitazone (10 μM). (a) Cells were immunostained with anti-HDAC6 (red) and anti-PPAR-γ (green) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( a , b ) IB3 - 1 were transfected with either HA - tagged human beclin 1 , empty vector , p62 siRNA or scrambled oligonucleotides followed by rosiglitazone ( 10 μM ) . ( b ) FRET of increase in PPAR - γ - Alexa 546 after N - CoR Cy5 photobleaching .

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(a, b) IB3-1 cells were transfected with either HA-tagged human beclin 1, empty vector, p62 siRNA or scrambled oligonucleotides followed by rosiglitazone (10 μM).(b) FRET analysis of increase in PPAR-γ-Alexa 546 fluorescence after N-CoR Cy5 photobleaching.


( c ) IB3 - 1 were transfected with either HA - tagged human beclin 1 or empty vector under nutrient starvation . were immunostained with anti - LC - 3 ( red ) anti - CFTR ( green ) . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(c) IB3-1 cells were transfected with either HA-tagged human beclin 1 or the empty vector under nutrient starvation. Cells were immunostained with anti-LC-3 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( d ) IB3 - 1 were cultured under nutrient starvation with cystamine ( 250 μM ) in presence or absence of PI ( 3 ) K inhibitor 3 - MA . Immunostaining was with anti - LAMP - 1 ( red ) anti - CFTR ( green ) . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(d) IB3-1 cells were cultured under nutrient starvation with cystamine (250 μM) in the presence or absence of the PI(3)K inhibitor 3-MA. Immunostaining was with anti-LAMP-1 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( e ) IB3 - 1 were transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in presence or absence of 250 μM cystamine under nutrient starvation . were immunostained with anti - p62 ( red ) anti - CFTR ( green ) antibodies . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(e) IB3-1 cells were transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of 250 μM cystamine under nutrient starvation. Cells were immunostained with anti-p62 (red) and anti-CFTR (green) antibodies. Yellow indicates co-localization. N, cell nucleus. Scale bar, 10 μm.


( f , g ) IB3 - 1 were transfected with 1 μg of pGFP - CFTRF508del or empty vector , in presence or absence of 250 μM cystamine . ( f ) were immunostained with anti - GM - 130 ( red ) anti - GFP ( green ) antibodies . Yellow indicates co - localization ( arrows ) . Scale bar , 10 μm . ( g ) FRET of GFP after HDAC6 Cy3 photobleaching . Each experiment was repeated three times . Quantitative measurement of co - localizations is shown in Supplementary Information , Fig . S9 .

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(f, g) IB3-1 cells were transfected with 1 μg of pGFP-CFTRF508del or the empty vector, in the presence or absence of 250 μM cystamine. (f) Cells were immunostained with anti-GM-130 (red) and anti-GFP (green) antibodies. Yellow indicates co-localization (arrows). Scale bar, 10 μm. (g) FRET analysis of GFP fluorescence after HDAC6 Cy3 photobleaching. Each experiment was repeated three times. Quantitative measurement of co-localizations is shown in Supplementary Information, Fig. S9.


( a - d ) IB3 - 1 transfected for 24 h at 37 ° C with 1 μg of pGFP - CFTRF508del or empty vector in presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides . ( a ) Left : CFTR revealed with anti - GFP antibody in IB3 - 1 lysate . Top arrow , - glycosylated form ( apparent Mr of band C about 170K , revealed as 210K with GFP tag ) ; bottom arrow , - glycosylated form ( apparent Mr of band B about 150K , revealed as 190K with GFP tag ) . Uncropped of blots are shown in Supplementary Information , Fig . S10 . Right : of bands B ( black ) C ( ) normalized to β - actin . Values are means ± s . d . for three independent experiments .

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(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides. (a) Left: CFTR revealed with anti-GFP primary antibody in IB3-1 cell lysate. Top arrow, complex-glycosylated form (apparent Mr of band C about 170K, revealed as 210K with GFP tag); bottom arrow, core-glycosylated form (apparent Mr of band B about 150K, revealed as 190K with GFP tag). Uncropped images of blots are shown in Supplementary Information, Fig. S10. Right: quantification of bands B (black) and C (white) normalized to β-actin levels. Values are means ± s.d. for three independent experiments.


( a - d ) IB3 - 1 transfected for 24 h at 37 ° C with 1 μg of pGFP - CFTRF508del or empty vector in presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides . ( b ) immunostained with an anti - GFP antibody under non - permeabilizing conditions . Serial confocal sections were collected from top to bottom of . ( c ) Side view ( X - Z ) of a z - stack of confocal taken in b . ( c ) Side view ( X - Z ) of a z - stack of confocal taken in b .

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(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(b) Cells immunostained with an anti-GFP antibody under non-permeabilizing conditions. Serial confocal sections were collected from top to bottom of cell. (c) Side view (X-Z) of a z-stack of confocal images taken in b. (c) Side view (X-Z) of a z-stack of confocal images taken in b.


( a - d ) IB3 - 1 transfected for 24 h at 37 ° C with 1 μg of pGFP - CFTRF508del or empty vector in presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides . ( d ) Surface biotinylation assays was performed with - impermeable sulpho - NHS - LC - biotin . were fractioned to obtain plasma fraction , biotinylated were precipitated with streptavidin beads . Immunoblot of GFP to reveal C form . E - cadherin β - actin were used as positive negative controls , respectively .

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(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(d) Surface biotinylation assays was performed with membrane-impermeable sulpho-NHS-LC-biotin. The cells were fractioned to obtain the plasma membrane fraction, and biotinylated proteins were precipitated with streptavidin beads. Immunoblot of GFP to reveal the C form. E-cadherin and β-actin were used as positive and negative controls, respectively.


( e ) IB3 - 1 transfected with either HA - tagged human beclin 1 or empty vector . Immunoblot of PIASy , TG2 , phospho - p42 / 44 PPAR - γ .

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(e) IB3-1 cells transfected with either HA-tagged human beclin 1 or empty vector. Immunoblot of PIASy, TG2, phospho-p42/44 and PPAR-γ proteins.


( f ) IB3 - 1 transfected with either 50 nM human p62 siRNA or scrambled oligonucleotides . Immunoblot of p62 phospho - p42 / 44 . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(f) IB3-1 cells transfected with either 50 nM human p62 siRNA or scrambled oligonucleotides. Immunoblot of p62 and phospho-p42/44. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( g ) IB3 - 1 transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in presence or absence of cystamine . Immunoblot of phospho - p42 / 44 .

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(g) IB3-1 cells transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. Immunoblot of phospho-p42/44.


( h ) IB3 - 1 transfected with either 50 nM humanbeclin 1 siRNA or 50 nM humanATG5 siRNA or scrambled oligonucleotides in presence or absence of cystamine . TNF - α secretion . Each bar represents mean ± s . d . for n = 3 experiments . Asterisk , P 0 . 01 ; ANOVA .

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(h) IB3-1 cells transfected with either 50 nM humanbeclin 1 siRNA or 50 nM humanATG5 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. TNF-α secretion. Each bar represents the mean ± s.d. for n = 3 experiments. Asterisk, P 0.01; ANOVA.


CF human nasal mucosae cultured with cystamine or NAC ( 4 - 10 in Supplementary Information , Table S1 ) ( a - c ) ( a ) Immunoblot of beclin 1 .

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CF human nasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) (a) Immunoblot of beclin 1.


CF humannasal mucosae cultured with cystamine or NAC ( 4 - 10 in Supplementary Information , Table S1 ) ( a - c ) in presence or absence of 3 - MA ( 6 - 10 in Supplementary Information , Table S1 ) ( b , c ) . ( b ) microscopy of LC3 - positive dots . Results represent means ± s . d . Asterisk , P 0 . 05 between groups of treatment ; ANOVA .

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CF humannasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) in the presence or absence of 3-MA (patients 6-10 in Supplementary Information, Table S1) (b, c). (b) Fluorescence microscopy quantification of LC3-positive dots. Results represent means ± s.d. Asterisk, P 0.05 between groups of treatment; ANOVA.


CF humannasal mucosae cultured with cystamine or NAC ( 4 - 10 in Supplementary Information , Table S1 ) ( a - c ) in presence or absence of 3 - MA ( 6 - 10 in Supplementary Information , Table S1 ) ( b , c ) . ( c ) Confocal microscopy of p62 . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm . Representative of five per group .

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CF humannasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) in the presence or absence of 3-MA (patients 6-10 in Supplementary Information, Table S1) (b, c). (c) Confocal microscopy micrographs of p62. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative of five patients per group.


( d - i ) CftrF508del mice treated with cystamine or PBS in presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( d ) Immunoblot of beclin 1 .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (d) Immunoblot of beclin 1.


( d - i ) CftrF508del mice treated with cystamine or PBS in presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( e ) of LC3 dots ( per mm2 of lung tissue ) . Means ± s . d . Asterisk , P 0 . 01 ; ANOVA .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (e) Quantification of LC3 dots (per mm2 of lung tissue). Means ± s.d. Asterisk, P 0.01; ANOVA.


( d - i ) CftrF508del mice treated with cystamine or PBS in presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( f ) Immunoblot of TG2 . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (f) Immunoblot of TG2. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( d - i ) CftrF508del mice treated with cystamine or PBS in presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( g ) Confocal microscopy of SUMO - 1 in lung tissues . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (g) Confocal microscopy of SUMO-1 in lung tissues. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm.


( d - i ) CftrF508del mice treated with cystamine or PBS in presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( h ) Number of CD68 + macrophages counted in 15 - 20 different randomly taken sections . Means ± s . d . per mm2 of lung tissue from seven mice in each group . Asterisk , P 0 . 01 ; ANOVA .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (h) Number of CD68+ macrophages counted in 15-20 different randomly taken sections. Means ± s.d. per mm2 of lung tissue from seven mice in each group. Asterisk, P 0.01; ANOVA.


( d - i ) CftrF508del mice treated with cystamine or PBS in presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( i ) MPO activity in lung homogenates ( expressed as 10 − 3 × optical density ( OD ) s − 1 ) . Means ± s . d . for lung tissues from seven mice in each group . Asterisk , P 0 . 05 ; ANOVA .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (i) MPO activity in lung homogenates (expressed as 10−3 × optical density (OD) s−1). Means ± s.d. for lung tissues from seven mice in each group. Asterisk, P 0.05; ANOVA.


( j - m ) CftrF508del mice ( n = 3 per group ) administered intranasally with a lentiviral vector encoding beclin 1 ( LV - beclin 1 ) or GFP ( LV - GFP ) . ( j ) TG2 Immunoblot in lung tissues . Uncropped of blots are shown in Supplementary Information , Fig . S10 .

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(j-m) CftrF508del mice (n = 3 per group) administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (j) TG2 Immunoblot in lung tissues. Uncropped images of blots are shown in Supplementary Information, Fig. S10.