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

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

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

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

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

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


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

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

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

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

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

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

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

O O O O O B-GENEPROD O B-GENEPROD O 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 O O O O O B-SMALL_MOLECULE O O O O O O O O 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 O O O O O O O O O O O O O O O O O O

(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 in absence presence of MMS . ChEC of G1 - synchronyzed released in absence or presence of 0 . 033 % MMS for indicated times . Data information : gel electrophoresis , content ( sorting ) of digestion profiles are shown . experiments were repeated twice with similar results .

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

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

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

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

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

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

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

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

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

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

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

(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 obtained with Imaris Surface software from high magnification of immunofluorescence against Iba - 1 showing representative microglial in Cx , Hip Cb of ASMko wt mice . Scale bars , 10 μm .

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

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

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O B-GENEPROD O B-ORGANISM O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O

(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 Filament Imaris software . ( I ) Mean ± branch point number per process process in iba1 + obtained with Imaris Filament software ( Student ´ s t - ) .

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-GENEPROD O O O O O O O O O O O O O O

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

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

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

O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-ORGANISM O O O O B-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 B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O B-GENEPROD O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

(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 wt mice with or without long - term PLX treatment ( n = 7 mice per group ) .

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

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


( H ) Immunofluorescence against PC marker Calbindin in mid posterior lobules of Cb in ASMko wt mice treated or not with PLX for 2 months . DAPI shows nuclei . Scale bar , 500 μm . ( I ) Mean ± number of Calbindin positive in anterior ( lobules I - V ) , middle ( lobules VI - VIII ) posterior zone ( lobules IX - X ) of Cb from 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 against CD16 / CD32 or Arg - 1 in Cb of ASMko mice ( red ) . Microglia were identified by F4 / 80 or Iba - 1 ( green ) . DAPI shows nuclei . Scale bar , 50 μm . ( B ) Mean ± number of Arg - 1 CD16 / CD32 positive microglia per in Cb from ASMko 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 ± of TNFa in Cb from ASMko wt mice measured by ELISA ( n = 7 mice per group , Student ´ s t - ) .

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

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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 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 against MBP in Cb of ASMko 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 ± MBP in 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 against MBP in ASMko mice treated or not with PLX for 2 months . arrows show MBP indicative of myelin debris . Scale bar , 50 μm . ( I ) Mean ± number of MBP per in different mouse groups ( n = 7 mice per group , Student ´ s t - ) .

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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 their 3D rendered replicates from cerebellar wt ASMko microglia identified by Iba - 1 of 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 its 3D rendered replicate of amoeboid microglia containing myelin debris ( arrowheads ) in ASMko cerebellum . orthogonal projection of this in lower panels confirms presence of myelin debris inside underneath plasma ( arrowhead ) . DAPI shows nuclei . Scale bars , 10 μm . ( L ) Mean ± of microglia showing internalised myelin debris in wt 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 Cb of ASMko mice showing accumulation of multillamelar vacuoles other lysosome related vesicles filling whole cytoplasm of these . Arrowhead identifies nuclei . Scale bar , 5 μm . On right , higher magnification showing a multillamelar ( arrowhead ) with 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 against Galectin3 - GFP in BV2 microglial 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 against Galectin3 - GFP in BV2 microglial 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 ± number of Galectin3 - GFP positive vesicles in BV2 microglial in different conditions ( n = 4 independent cultures , > 50 per culture , Student ´ s t - ) .

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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 in BMDMs from wt 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 GAPDH in fractions of wt ASMko BMDMs extracted with digitonin ( left ) . Mean ± pro - CathB CathB in fractions ( right ) ( n = 2 cultures per genotype , Student ´ s t - ) .

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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 against Iba - 1 ( green ) CathB ( red ) in Cb of ASMko wt mice . Arrowheads show double positive . Scale bar , 50 μm . Below , magnified of selected areas . Scale bar , 20 μm . ( H ) Immunofluorescence against CathB ( green ) , Arg - 1 ( red , anti - inflammatory microglia ) F4 / 80 ( yellow , microglia ) in Cb of ASMko mice . DAPI shows nuclei . Scale bar , 50 μm . Below , magnified of selected areas . Scale bar , 20 μm . ( I ) Table showing mean ± of microglia presenting CathB ( CathB + / Iba - 1 + ) , number of CathB positive microglia co - expressing Arg - 1 ( CathB + / Arg - 1 + ) in Cb of ASMko wt mice ( n = 5 mice , > 10 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 ( for both precursor ( pro - CathB ) - mature forms ) in culture media from ASMko wt BMDMs . of Ponceau - S is shown as loading control . ( B ) Mean ± fold increase in CathB in culture media from wt ASMko BMDMs ( n = 5 independent cultures , Student ´ s t - ) .

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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 against LAMP - 1 ( yellow ) Iba - 1 ( red ) in Cb of ASMko wt mice . Scale bar , 50 μm , zoomed 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 against LAMP - 1 in non - permeabilised BMDMs from ASMko 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 ± of LAMP - 1 on surface of ASMko wt BMDMs ( n = 4 independent cultures , > 40 per culture , Student ´ s t - ) .

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

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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 Flotillin1 in secreted exosome fractions from ASMko wt BMDMs . ( H ) Western blot of CathB in exosome depleted culture media from wt 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 ± MTT absorbance reflecting viability in neurons from wt mice treated with conditioned media from ASMko wt BMDMs in presence or absence of 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 against Iba - 1 ( green ) CathB ( red ) in lobes of Cb sensitive or resistant to Purkinje . Asterisks indicate Purkinje in 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 ± of CathB positive microglia in resistant sensitive Cb lobules of ASMko mice ( n = 5 mice , χ2 ) .

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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 β - Actin in cerebellar extracts of wt ASMko mice treated or not with PLX . ( M ) Mean ± of CathB normalized by β - Actin in cerebellar extracts of wt 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 ( for both precursor ( pro - CathB ) - mature forms ) in culture media from ASMko 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 , 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 ± increase in pro - CathB ( left ) CathB ( right ) in culture media from cerebellar organotypic cultures from wt 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 ± of ASMko wt mice treated or not with Ca074Me at 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 Rotarod of ASMko wt mice treated or not with Ca074Me for 1 month expressed as mean ± time spent in 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 Rotarod of ASMko wt mice treated or not with Ca074Me for 1 month expressed as mean ± time of 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 against PC marker Calbindin in posterior lobules of Cb in ASMko wt mice treated or not with Ca074Me for 1 month . DAPI shows 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 ± number of Calbindin positive in posterior zone ( lobules IX - X ) of Cb from 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 against Iba - 1 in Cx , Hip Cb of 2 month - old Npc1nmf164 wt mice . DAPI shows 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 obtained with Imaris Surface software from high magnification z - stack of immunofluorescence against Iba - 1 showing representative microglial in Cx , Hip Cb of Npc1nmf164 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 ± number ( upper panel ) ( lower panel ) of Iba - 1 positive in different brain regions from Npc1nmf164 wt mice ( n = 5 mice per group , > 30 per mouse , Student ´ s t - ) .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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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 mean of at least ten epidermal thickness measurements for each ear ; represents mean value .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( B ) Delay in distal colonic transit time .

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(B) Delay in distal colonic transit time.


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

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


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

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


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

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


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

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


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

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


Diameter analyses of ENCC - derived neurospheres ( F ) ,

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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.