EMBO/SourceData huggingface dataset:
Figure 3(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.
Figure 4fluorescence 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).
Figure 6(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.
Figure 7(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.
Figure 8(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.
Figure 1(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.(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.(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).
Figure 2(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).(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).(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).
Figure 3(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).(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).(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).(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.(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).
Figure 4(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.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).(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).(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).
Figure 5(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.(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).(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.(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.(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). (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).
Figure 6(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)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.(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).
Figure 7(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.(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).(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.(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.(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.
Figure 1Representative 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.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)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).
Figure 2Changes 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.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).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).
Figure 3Cleavage 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).Representative histological sections of ear tissue stained with hematoxylin and eosinmeasurements 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).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).
Figure 1Comparison 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.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,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.
Figure 2Comparison 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.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.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.