( H ) High magnification of immunofluorescence staining against MBP in ASMko mice treated or not with PLX for 2 months . White arrows show MBP aggregates indicative of myelin debris . Scale bar , 50 μm . ( I ) Mean ± SEM number of MBP aggregates per cell area in the different mouse groups ( n = 7 mice per group , Student ´ s t - test ) .

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(H) High magnification of immunofluorescence staining against MBP in ASMko mice treated or not with PLX for 2 months. White arrows show MBP aggregates indicative of myelin debris. Scale bar, 50 μm. (I) Mean ± SEM number of MBP aggregates per cell area in the different mouse groups (n = 7 mice per group, Student´s t-test).


( J ) High magnification images and their 3D rendered replicates from cerebellar wt and ASMko microglia identified by Iba - 1 and of the myelin marker MBP . Scale bars , 10 μm .

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(J) High magnification images and their 3D rendered replicates from cerebellar wt and ASMko microglia identified by Iba-1 and of the myelin marker MBP. Scale bars, 10 μm.


( K ) Amplified image and its 3D rendered replicate of amoeboid microglia containing myelin debris ( arrowheads ) in the ASMko cerebellum . The orthogonal projection of this cell in the lower panels confirms the presence of the myelin debris inside the cell and underneath the plasma membrane ( arrowhead ) . DAPI staining shows cell nuclei . Scale bars , 10 μm . ( L ) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and ASMko mice ( n = 7 mice per group ) .

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(K) Amplified image and its 3D rendered replicate of amoeboid microglia containing myelin debris (arrowheads) in the ASMko cerebellum. The orthogonal projection of this cell in the lower panels confirms the presence of the myelin debris inside the cell and underneath the plasma membrane (arrowhead). DAPI staining shows cell nuclei. Scale bars, 10 μm. (L) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and ASMko mice (n = 7 mice per group).


( A ) Transmission electron microscopy image of microglia in the Cb of ASMko mice showing accumulation of multillamelar vacuoles and other lysosome related vesicles filling the whole cytoplasm of these cells . Arrowhead identifies the nuclei . Scale bar , 5 μm . On the right , higher magnification showing a multillamelar body ( arrowhead ) with the characteristic concentric lamellar inclusions . Scale bar , 0 . 5 μm .

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(A) Transmission electron microscopy image of microglia in the Cb of ASMko mice showing accumulation of multillamelar vacuoles and other lysosome related vesicles filling the whole cytoplasm of these cells. Arrowhead identifies the nuclei. Scale bar, 5 μm. On the right, higher magnification showing a multillamelar body (arrowhead) with the characteristic concentric lamellar inclusions. Scale bar, 0.5 μm.


Immunofluorescence staining against Galectin3 - GFP in BV2 microglial cells treated with or without SM ( B ) Scale bar , 10 μm .

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Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without SM (B) Scale bar, 10 μm.


Immunofluorescence staining against Galectin3 - GFP in BV2 microglial cells treated with or without Siramesine ( C ) . Scale bar , 10 μm .

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Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without Siramesine (C). Scale bar, 10 μm.


( D ) Mean ± SEM number of Galectin3 - GFP positive vesicles in BV2 microglial cells in the different conditions ( n = 4 independent cultures , > 50 cells per culture , Student ´ s t - test ) .

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(D) Mean ± SEM number of Galectin3-GFP positive vesicles in BV2 microglial cells in the different conditions (n = 4 independent cultures, >50 cells per culture, Student´s t-test).


( E ) Lysotracker - Red staining in BMDMs from wt and ASMko mice . Scale bar , 10 μm .

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(E) Lysotracker-Red staining in BMDMs from wt and ASMko mice. Scale bar, 10 μm.


( F ) Western blot of CathB levels and the cytosolic protein GAPDH in the cellular and cytosolic fractions of wt and ASMko BMDMs extracted with digitonin ( left ) . Mean ± SEM pro - CathB and CathB levels in the cytosolic fractions ( right ) ( n = 2 cultures per genotype , Student ´ s t - test ) .

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(F) Western blot of CathB levels and the cytosolic protein GAPDH in the cellular and cytosolic fractions of wt and ASMko BMDMs extracted with digitonin (left). Mean ± SEM pro-CathB and CathB levels in the cytosolic fractions (right) (n=2 cultures per genotype, Student´s t-test).


( G ) Immunofluorescence staining against Iba - 1 ( green ) and CathB ( red ) in Cb of ASMko and wt mice . Arrowheads show double positive cells . Scale bar , 50 μm . Below , magnified images of the selected areas . Scale bar , 20 μm . ( H ) Immunofluorescence staining against CathB ( green ) , Arg - 1 ( red , anti - inflammatory microglia ) and F4 / 80 ( yellow , all microglia ) in Cb of ASMko mice . DAPI staining shows cell nuclei . Scale bar , 50 μm . Below , magnified images of the selected areas . Scale bar , 20 μm . ( I ) Table showing mean ± SEM percentage of microglia presenting CathB staining ( CathB + / Iba - 1 + ) , and the number of CathB positive microglia co - expressing Arg - 1 ( CathB + / Arg - 1 + ) in Cb of ASMko and wt mice ( n = 5 mice , > 10 cells per mouse ) .

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(G) Immunofluorescence staining against Iba-1 (green) and CathB (red) in Cb of ASMko and wt mice. Arrowheads show double positive cells. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (H) Immunofluorescence staining against CathB (green), Arg-1 (red, anti-inflammatory microglia) and F4/80 (yellow, all microglia) in Cb of ASMko mice. DAPI staining shows cell nuclei. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (I) Table showing mean ± SEM percentage of microglia presenting CathB staining (CathB +/Iba-1+), and the number of CathB positive microglia co-expressing Arg-1 (CathB+/Arg-1+) in Cb of ASMko and wt mice (n = 5 mice, >10 cells per mouse).


( A ) Western blot of CathB levels ( for both the precursor ( pro - CathB ) and the cleaved - mature forms ) in the culture media from ASMko and wt BMDMs . Staining of Ponceau - S is shown as loading control . ( B ) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs ( n = 5 independent cultures , Student ´ s t - test ) .

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(A) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt BMDMs. Staining of Ponceau-S is shown as loading control. (B) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs (n = 5 independent cultures, Student´s t-test).


( C ) Immunofluorescence staining against LAMP - 1 ( yellow ) and Iba - 1 ( red ) in Cb of ASMko and wt mice . Scale bar , 50 μm , zoomed images 20 μm .

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(C) Immunofluorescence staining against LAMP-1 (yellow) and Iba-1 (red) in Cb of ASMko and wt mice. Scale bar, 50 μm, zoomed images 20 μm.


( D ) Immunofluorescence staining against LAMP - 1 in non - permeabilised BMDMs from ASMko and wt mice . Scale bar , 50 μm .

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(D) Immunofluorescence staining against LAMP-1 in non-permeabilised BMDMs from ASMko and wt mice. Scale bar, 50 μm.


( E ) Mean ± SEM of LAMP - 1 levels on the surface of ASMko and wt BMDMs ( n = 4 independent cultures , > 40 cells per culture , Student ´ s t - test ) .

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(E) Mean ± SEM of LAMP-1 levels on the surface of ASMko and wt BMDMs (n = 4 independent cultures, >40 cells per culture, Student´s t-test).


( F ) Mean ± SEM microvesicle and exosome numbers in the culture media from ASMko and wt BMDMs ( n = 6 independent cultures , Student ´ s t - test ) .

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(F) Mean ± SEM microvesicle and exosome numbers in the culture media from ASMko and wt BMDMs (n = 6 independent cultures, Student´s t-test).


( G ) Western blot of CathB and Flotillin1 levels in the secreted exosome fractions from ASMko and wt BMDMs . ( H ) Western blot of CathB levels in the exosome depleted culture media from wt and ASMko BMDMs .

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(G) Western blot of CathB and Flotillin1 levels in the secreted exosome fractions from ASMko and wt BMDMs. (H) Western blot of CathB levels in the exosome depleted culture media from wt and ASMko BMDMs.


( I ) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 ( n = 4 independent cultures Two - way Anova , Bonferroni post hoc ) .

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(I) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 (n = 4 independent cultures Two-way Anova, Bonferroni post hoc).


( J ) Immunofluorescence staining against Iba - 1 ( green ) and CathB ( red ) in lobes of the Cb sensitive or resistant to Purkinje cell death . Asterisks indicate Purkinje cells in the resistant lobes . Scale bar , 50 μm .

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(J) Immunofluorescence staining against Iba-1 (green) and CathB (red) in lobes of the Cb sensitive or resistant to Purkinje cell death. Asterisks indicate Purkinje cells in the resistant lobes. Scale bar, 50 μm.


( K ) Mean ± SEM percentage of CathB positive microglia in resistant and sensitive Cb lobules of ASMko mice ( n = 5 mice , χ2 test ) .

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(K) Mean ± SEM percentage of CathB positive microglia in resistant and sensitive Cb lobules of ASMko mice (n = 5 mice, χ2 test).


( L ) Western blot of Cath B and β - Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX . ( M ) Mean ± SEM of CathB levels normalized by β - Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX ( n = 6 mice Two - way Anova , Bonferroni post hoc ) .

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(L) Western blot of Cath B and β-Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX. (M) Mean ± SEM of CathB levels normalized by β-Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX (n= 6 mice Two-way Anova, Bonferroni post hoc).


( O ) Western blot of CathB levels ( for both the precursor ( pro - CathB ) and the cleaved - mature forms ) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle ( veh ) or PLX ( PLX ) . Below , Western blots of organotypic culture lysates against Iba - 1 , to confirm microglia depletion , and against GAPDH as loading control .

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(O) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle (veh) or PLX (PLX). Below, Western blots of organotypic culture lysates against Iba-1, to confirm microglia depletion, and against GAPDH as loading control.


( P ) Mean ± SEM increase in pro - CathB ( left ) and CathB ( right ) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX ( n = 3 mice per group Two - way Anova , Bonferroni post hoc ) .

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(P) Mean ± SEM increase in pro-CathB (left) and CathB (right) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX (n = 3 mice per group Two-way Anova, Bonferroni post hoc).


( A ) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment ( n = 7 mice per group ) .

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(A) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment (n = 7 mice per group).


Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials ( B )

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Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials (B)


Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time of the four trials ( C )

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Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time of the four trials (C)


( D ) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month . DAPI staining shows cell nuclei . Scale bar , 200 μm .

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(D) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month. DAPI staining shows cell nuclei. Scale bar, 200 μm.


( E ) Mean ± SEM number of Calbindin positive cells in the posterior zone ( lobules IX - X ) of the Cb from the different mouse groups ( n = 7 mice per group Two - way Anova , Bonferroni post hoc ) .

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(E) Mean ± SEM number of Calbindin positive cells in the posterior zone (lobules IX-X) of the Cb from the different mouse groups (n= 7 mice per group Two-way Anova, Bonferroni post hoc).


( A ) Immunofluorescence staining against Iba - 1 in Cx , Hip and Cb of 2 month - old Npc1nmf164 and wt mice . DAPI staining shows cell nuclei . Scale bars , 30 μm .

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(A) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 2 month-old Npc1nmf164 and wt mice. DAPI staining shows cell nuclei. Scale bars, 30 μm.


( B ) 3D rendered images obtained with Imaris Surface software from high magnification z - stack images of immunofluorescence staining against Iba - 1 showing representative microglial morphology in Cx , Hip and Cb of Npc1nmf164 and wt mice . Scale bars , 10 μm .

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(B) 3D rendered images obtained with Imaris Surface software from high magnification z-stack images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of Npc1nmf164 and wt mice. Scale bars, 10 μm.


( 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 ± SEM percentage of microglia showing internalised myelin debris in wt and 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 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 .

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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 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 .

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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 protein levels in skin and 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 images of K5creTg / + CARD14E138A + / + and 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 + / + and 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.


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 )

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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 and skin tissue of Malt1fl / fl K5cre + / + CARD14E138A + / + and 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 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 ) .

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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 cells .

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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 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 .

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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.


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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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).


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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 the mean of at least ten epidermal thickness measurements for each ear ; the line represents the 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 and 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).


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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ,

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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

O O O O O O O O O B-TISSUE O O B-DISEASE B-ORGANISM O B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

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 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 .

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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 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 .

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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 the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and 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 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 .

O O O O O O O O O B-TISSUE O O B-DISEASE B-ORGANISM O B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-ORGANISM O O O B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

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 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 ) .

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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 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 .

O O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-DISEASE O B-DISEASE O O O B-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-TISSUE O O O O O O O O O B-DISEASE O B-DISEASE O O O B-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O

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 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 .

O O B-TISSUE O O O O O O O O O B-DISEASE O B-DISEASE O O O B-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-TISSUE O O O O B-EXP_ASSAY O

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 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 .

O O B-TISSUE O O O O O O O O O B-DISEASE O B-DISEASE O O O B-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O

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

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

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(B) Delay in distal colonic transit time.


( 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 .

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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 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 .

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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 + and Tuj1 + / GFP + cells ( F ) and Tuj1 + / GFP - cells ( 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 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 ) .

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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 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 ) .

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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 the ENCC - derived neurospheres ( F ) ,

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


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 .

B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O

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 assay show that MEK1 / 2 inhibitor U0126 suppressed SCFA - induced ENCC proliferation and migration .

B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O

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


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 .

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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 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 .

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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 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 .

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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 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 .

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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 the number of LC3puncta per cell , as described in E ( mean ± s . d . ; n > 100 cells 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 cells 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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O 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 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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O 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 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 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 .

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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 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 .

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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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

C 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 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 .

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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 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 .

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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 cells 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 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 .

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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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O 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 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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C 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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 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 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 .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O 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-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O 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 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 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 ) .

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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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_LINE O O O O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O 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-CELL_LINE O O O O

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

(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 and IB3 - 1 cells ( d ) and IB3 - 1 cells ( e ) after incubation with E64d and 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 and IB3 - 1 cells . Uncropped images 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 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 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( 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 .

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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 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 .

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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 ) 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 .

O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O B-ORGANISM O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O

(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 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 .

O O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O

(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 and beclin 1 . Uncropped images 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 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 .

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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 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 .

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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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) 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 and LC3 . ( i ) Immunoblot of p62 . Uncropped images 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 and 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.