( E ) Fold change expression of NRF2 - target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset ( n = 6 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - test .

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(E) Fold change expression of NRF2-target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset (n = 6/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( H ) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction . Asterix indicating non - specific band ( n = 14 - 18 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - test .

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(H) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction. Asterix indicating non-specific band (n = 14-18/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( B ) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum - starved medium . Secretion of IL - 10 and from mesenteric ( left panel ) and gonadal adipose tissues ( right panel ) was measured by ELISA . Shapes identify individual experiments ( n = 5 - 15 / group ) . Data are represented as mean ± s . e . m . ( B ) Two - Way ANOVA with regression for experiment .

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(B) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum-starved medium. Secretion of IL-10 and from mesenteric (left panel) and gonadal adipose tissues (right panel) was measured by ELISA. Shapes identify individual experiments (n = 5-15/group). Data are represented as mean ± s.e.m. (B) Two-Way ANOVA with regression for experiment.


( F ) Serum cytokines upon DSS - induced colitis at day 7 post - induction ( n = 17 - 23 / group ) . Data are represented as mean ± s . e . m . Two - Way ANOVA .

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(F) Serum cytokines upon DSS-induced colitis at day 7 post-induction (n = 17-23/group). Data are represented as mean ± s.e.m. Two-Way ANOVA.


( A ) Quantification of Il10 transcript levels in RAW264 . 7 upon stimulation to epoxy fatty acids ( n = 3 / group ) . ( B ) Quantification of IL - 10 protein levels in the supernatant of RAW264 . 7 upon stimulation to epoxy fatty acids ( n = 3 / group ) . Data are represented as mean ± s . e . m . ( A , B ) One - Way ANOVA .

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(A) Quantification of Il10 transcript levels in RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). (B) Quantification of IL-10 protein levels in the supernatant of RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). Data are represented as mean ± s.e.m. (A,B) One-Way ANOVA.


( C - G ) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course ( right panels ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour ( 60 + ) , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52 - YFP foci ( 60 - ) . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( left panels ) . Cell cycle progression was determined by cell sorting . The mean and SEM of 3 ( wild type , rad18 ∆ , mms2 ∆ , rad51 ∆ , rad57 ∆ , rad54 ∆ ) , 4 ( pol30 - K164R ) and 5 ( rad5 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where three asterisks represent p - values < 0 . 001 .

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(C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.


( C - E ) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course ( D and E , right panel ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( C and E , left panel ) . Data information : The mean and SEM of and 3 - 12 12 ( wild type ) , 6 ( rev1 ∆ , rev3 ∆ , rad30 ∆ , rev1 ∆ rad30 ∆ , rev3 ∆ rad30 ∆ ) , and 3 ( rev1 ∆ rev3 ∆ and rev1 ∆ rev3 ∆ rad30 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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(C-E) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel). Data information: The mean and SEM of and 3-12 12 (wild type), 6 (rev1∆, rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆ rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( C - D ) ssDNA repair efficiency of the rad5 ∆ and rev1 ∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course ( right panels ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( left panels ) . Data information : The mean and SEM of independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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(C-D) ssDNA repair efficiency of the rad5∆ and rev1∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Data information: The mean and SEM of independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


fluorescence signal of RPA foci per cell ( E ) at times 1 or 2 ( peak ) and 7 hours from the time course shown in ( B ) . Data information : The mean and SEM of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . One , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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fluorescence signal of RPA foci per cell (E) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( F ) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1 - YFP at the ARS305 replication origin . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium . The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release . RPA enrichment was calculated as the ratio between immunoprecipitate and input values . All values were normalized to the wild type at 7 hours , taken as 1 . Data information : The mean and SEM of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . One , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively . In ( F ) all values were significant relative to the untagged controls ( not shown for clarity ) .

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(F) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release. RPA enrichment was calculated as the ratio between immunoprecipitate and input values. All values were normalized to the wild type at 7 hours, taken as 1. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).


Effect of the lack of HR factors in spontaneous and MMS - induced mutagenesis . The frequency of spontaneous and MMS - induced forward mutagenesis at the CAN1 locus was determined in mid - log phase cell cultures before ( spontaneous mutagenesis ) and after treatment with 0 . 01 % MMS for 4 hours ( DNA damage - induced ) . Thus , the frequency of DNA damage - induced mutagenesis includes the mutants that arose spontaneously before MMS addition . The frequency of mutagenesis was determined at 0 . 01 % MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine - resistant cells . Survival ( % ) after 4 hours in MMS relative to the wild type ( taken as 100 ) is shown below the genotypes . The mean and SEM of 6 ( wild type ) , 9 ( rad54 ∆ ) and 3 ( rest ) independent fluctuation tests are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test ( one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively ) . Only the statistical analysis of the effect of rad52 ∆ , rad57 ∆ and rad51 ∆ on rad54 ∆ - mediated mutagenesis is included for clarity . All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS .

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Effect of the lack of HR factors in spontaneous and MMS-induced mutagenesis. The frequency of spontaneous and MMS-induced forward mutagenesis at the CAN1 locus was determined in mid-log phase cell cultures before (spontaneous mutagenesis) and after treatment with 0.01% MMS for 4 hours (DNA damage-induced). Thus, the frequency of DNA damage-induced mutagenesis includes the mutants that arose spontaneously before MMS addition. The frequency of mutagenesis was determined at 0.01% MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine-resistant cells. Survival (%) after 4 hours in MMS relative to the wild type (taken as 100) is shown below the genotypes. The mean and SEM of 6 (wild type), 9 (rad54∆) and 3 (rest) independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test (one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively). Only the statistical analysis of the effect of rad52∆, rad57∆ and rad51∆ on rad54∆-mediated mutagenesis is included for clarity. All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS.


( A ) Effect of the rad54 ∆ and rad52 ∆ mutations in spontaneous and UV - induced uSCE . The frequency of spontaneous and UV - induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions ( SMM and SMM without histidine to calculate total and recombinant cells , respectively ) . The mean and SEM of 3 - 5 independent fluctuation tests are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . Two and three asterisks represent p - values < 0 . 01 and < 0 . 001 , respectively .

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(A) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced uSCE. The frequency of spontaneous and UV-induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM without histidine to calculate total and recombinant cells, respectively). The mean and SEM of 3-5 independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. Two and three asterisks represent p-values <0.01 and <0.001, respectively.


( B ) Rad6 binds to DNA in the absence and presence of MMS . ChEC analysis of G1 - synchronyzed cells released in the absence or the presence of 0 . 033 % MMS for the indicated times . Data information : DNA gel electrophoresis , DNA content ( cell sorting ) and quantification of DNA digestion profiles are shown . The experiments were repeated twice with similar results .

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(B) Rad6 binds to DNA in the absence and presence of MMS. ChEC analysis of G1-synchronyzed cells released in the absence or the presence of 0.033% MMS for the indicated times. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( C - F ) Rad6 binding to DNA depends on Rad18 , Rad52 ( C ) , Rad51 ( D ) , Rad57 ( E ) but not Rad54 ( F ) as determined by ChEC analyses of asynchronous cell cultures without or with 0 . 05 % MMS for 2 hours . Data information : DNA gel electrophoresis , DNA content ( cell sorting ) and quantification of DNA digestion profiles are shown . The experiments were repeated twice with similar results .

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(C-F) Rad6 binding to DNA depends on Rad18, Rad52 (C), Rad51 (D), Rad57 (E) but not Rad54 (F) as determined by ChEC analyses of asynchronous cell cultures without or with 0.05% MMS for 2 hours. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( A - D ) Effect of rad52 ∆ , rad51 ∆ ( A ) , rad57 ∆ ( B ) and rad54 ∆ ( C ) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0 . 025 % MMS . Cell cycle progression was followed by cell sorting analysis and western blot against Clb2 . Pgk1 was used as loading control . The amounts of mono - and polyubiquitylated PCNA were normalized to the total amount of PCNA . For each time point , rad mutant values were relativized to those of the wild type , taken as 1 . The mean and SEM of 3 independent experiments are shown ( D ) . An asterisk indicates a mean significantly different than 1 , according to a One - sample t - test .

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(A-D) Effect of rad52∆, rad51∆ (A), rad57∆ (B) and rad54∆ (C) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0.025% MMS. Cell cycle progression was followed by cell sorting analysis and western blot against Clb2. Pgk1 was used as loading control. The amounts of mono- and polyubiquitylated PCNA were normalized to the total amount of PCNA. For each time point, rad mutant values were relativized to those of the wild type, taken as 1. The mean and SEM of 3 independent experiments are shown (D). An asterisk indicates a mean significantly different than 1, according to a One-sample t-test.


( A ) Immunofluorescence staining against Iba - 1 in cortex ( Cx ) , hippocampus ( Hip ) and cerebellum ( Cb ) of NPA affected and control 3 year - old children . Scale bars , 50 μm . ( B ) Magnified image showing ramified ( left ) versus amoeboid ( right ) morphology in control and NPA microglia . Scale bars , 10 μm . ( C ) Mean ± SEM area of Iba - 1 positive cells in different brain regions from a NPA affected and a control child ( n = 20 cells , Student ´ s t - test ) .

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(A) Immunofluorescence staining against Iba-1 in cortex (Cx), hippocampus (Hip) and cerebellum (Cb) of NPA affected and control 3 year-old children. Scale bars, 50 μm. (B) Magnified image showing ramified (left) versus amoeboid (right) morphology in control and NPA microglia. Scale bars, 10 μm. (C) Mean ± SEM area of Iba-1 positive cells in different brain regions from a NPA affected and a control child (n = 20 cells, Student´s t-test).


( D ) Immunofluorescence staining against Iba - 1 in Cx , Hip and Cb of 3 month - old ASMko and wt mice . DAPI staining shows cell nuclei . Scale bars , 50 μm .

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(D) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 3 month-old ASMko and wt mice. DAPI staining shows cell nuclei. Scale bars, 50 μm.


( E ) Mean ± SEM number of Iba - 1 positive cells in different brain regions from ASMko and wt mice ( n = 5 mice per group , Student ´ s t - test ) .

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(E) Mean ± SEM number of Iba-1 positive cells in different brain regions from ASMko and wt mice (n = 5 mice per group, Student´s t-test).


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

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


( G ) Mean ± SEM area of Iba - 1 positive cells in the different brain regions from ASMko and wt mice ( n = 5 mice per group , > 10 cells per mouse , Student ´ s t - test ) . Scale bars , 15 μm .

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(G) Mean ± SEM area of Iba-1 positive cells in the different brain regions from ASMko and wt mice (n = 5 mice per group, >10 cells per mouse, Student´s t-test). Scale bars, 15 μm.


( H ) Example of projection detection by the Filament Imaris software . ( I ) Mean ± SEM branch point number per process and process length in iba1 + cells obtained with the Imaris Filament software ( Student ´ s t - test ) .

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(H) Example of projection detection by the Filament Imaris software. (I) Mean ± SEM branch point number per process and process length in iba1+ cells obtained with the Imaris Filament software (Student´s t-test).


( A ) Immunofluorescence staining against Iba - 1 in Cb of ASMko and wt mice treated or not with PLX for 2 months . DAPI staining shows cell nuclei . Scale bar , 100 μm . ( B ) Magnified images ( from A ) showing ramified ( left ) versus amoeboid ( right ) morphology in wt and ASMko microglia treated or not with PLX . Scale bars , 30 μm . ( C ) Mean ± SEM number of Iba - 1 positive cells in the Cb of the different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(A) Immunofluorescence staining against Iba-1 in Cb of ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 100 μm. (B) Magnified images (from A) showing ramified (left) versus amoeboid (right) morphology in wt and ASMko microglia treated or not with PLX. Scale bars, 30 μm. (C) Mean ± SEM number of Iba-1 positive cells in the Cb of the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).


( D , E ) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials ( D ) or the mean ± SEM time of the four trials ( E ) ( n = 7 mice per group , Two - way Anova , Games - Howell ( D ) and Bonferroni ( E ) post hoc ) .

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(D, E) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials (D) or the mean ± SEM time of the four trials (E) (n = 7 mice per group, Two-way Anova, Games-Howell (D) and Bonferroni (E) post hoc).


( F ) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long - term treatment ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(F) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long-term treatment (n= 7 mice per group, Two-way Anova, Bonferroni post hoc).


( G ) Survival curve of ASMko and wt mice with or without long - term PLX treatment ( n = 7 mice per group ) .

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(G) Survival curve of ASMko and wt mice with or without long-term PLX treatment (n = 7 mice per group).


( H ) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months . DAPI staining shows cell nuclei . Scale bar , 500 μm . ( I ) Mean ± SEM number of Calbindin positive cells in the anterior ( lobules I - V ) , middle ( lobules VI - VIII ) and posterior zone ( lobules IX - X ) of the Cb from the different mouse groups ( n = 7 mouse per group , Two - way Anova , Bonferroni post hoc ) .

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(H) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 500 μm. (I) Mean ± SEM number of Calbindin positive cells in the anterior (lobules I-V), middle (lobules VI-VIII) and posterior zone (lobules IX-X) of the Cb from the different mouse groups (n = 7 mouse per group, Two-way Anova, Bonferroni post hoc).


( A ) Immunofluorescence staining against CD16 / CD32 or Arg - 1 in Cb of ASMko mice ( red ) . Microglia were identified by F4 / 80 or Iba - 1 staining ( green ) . DAPI staining shows cell nuclei . Scale bar , 50 μm . ( B ) Mean ± SEM number of Arg - 1 and CD16 / CD32 positive microglia per area in Cb from ASMko and wt mice ( n = 6 mice per group ) .

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(A) Immunofluorescence staining against CD16/CD32 or Arg-1 in Cb of ASMko mice (red). Microglia were identified by F4/80 or Iba-1 staining (green). DAPI staining shows cell nuclei. Scale bar, 50 μm. (B) Mean ± SEM number of Arg-1 and CD16/CD32 positive microglia per area in Cb from ASMko and wt mice (n = 6 mice per group).


( C ) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA ( n = 7 mice per group , Student ´ s t - test ) .

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(C) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA (n = 7 mice per group, Student´s t-test).


( D ) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT - PCR ( n = 7 mice per group , Student ´ s t - test ) .

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(D) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT-PCR (n = 7 mice per group, Student´s t-test).


( E ) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone ( n = 8 mice per group ) .

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(E) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone (n = 8 mice per group).


( F ) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months . Scale bar , 50 μm .

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(F) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months. Scale bar, 50 μm.


( G ) Mean ± SEM MBP intensity in the different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(G) Mean ± SEM MBP intensity in the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).


( 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).


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 .

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 ) .

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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

(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 .

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

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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 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 .

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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.