EMBO/SourceData huggingface dataset:
Figure 5(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.(C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.(D) Kidney ILC2s isolated from normal IL-10 reporter C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for six days. Proportion of ILC210 was assessed by flow cytometry. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.(E) The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.(F) Kidney ILC2s isolated from C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for 30 minutes. The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry. p-STAT5 (red lines) and isotype controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=6 per group) and an unpaired t-test was performed; ***P<0.001. MFI, mean fluorescence intensity.(G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.
Figure 6(A) ILC210 and non-ILC210 were isolated from ex vivo-expanded kidney ILC2 by flow sorting. ILC210 were transfected with control (ILC210-C) or IL-10 CRISPR-Cas9 (ILC210-IL-10). IL-10 was measured in culture supernatant of ILC210-C and ILC210-IL-10 via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.(C) Transfused ILC2s (CFSE labeled, Green) were observed in islet graft at day 5 post islet transplantation. Scale bar = 50 µm. The numbers of CFSE-labeled ILC2s in the islet graft were counted. Data shown are the mean ± SEM and a one-way ANOVA was performed (n=4 per group). NS, not significant.(D) The mRNA expression of IL-10 in islet grafts at day 5 post islet transplantation was examined by qPCR. Data shown are the mean ± SEM (n=4 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.(E) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.(F) CD4+ T cells isolated from C57BL/6 splenocytes were cultured with irradiated splenocytes derived from BALB/c in the presence of ILC210 at the indicated ratios for 4 days. CD4+ T cells proliferation was assessed using [3H]-thymidine incorporation assays. Data shown are the mean ± SEM (n=4-6 per group).(G) Neutralizing anti-IL-10 antibodies or genetic ablation of IL-10 were used to block the effect of ILC210 on CD4+ T cells proliferation. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed. ***P<0.001.
Figure 7(B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.(C) To track ILC210 in vivo, CD45.2+ ILC210 were co-transplanted with islets in diabetic CD45.1+ C57BL/6 mice. Representative FACS analysis showing the proportion of locally transplanted ILC210 (CD45.2+ST2+) in the total CD45+ cell compartment from islet graft at day 5 and 80 post-islet transplantation or at the day when grafts were considered rejected.(D,E) Proportion and numbers of locally transplanted CD45.2+ST2+ ILC210 in islet grafts of islet transplant mice over time. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed, **P<0.01, ***P<0.001.(F) The expression of IL-10-GFP was examined in locally transplanted CD45.2+ST2+ ILC210 by flow cytometry. IL-10-GFP (red lines) and controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=4-5 per group) and a one-way ANOVA was performed; ***P<0.001. MFI, mean fluorescence intensity.
Figure 1B. ST2 cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in five independent experiments are shown.C. The stoichiometry of IL-17RSC calculated as the ratio between iBAQ intensities of individual IL-17RSC components to iBAQ intensity of IL-17RC. The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced. Mean from five performed MS experiments is shown, the statistical significance was determined using unpaired two‐tailed Student's t‐test.D. ST2 cells were stimulated with IL-17 (500 ng/ml), TNF (50 ng/ml), or IL-1α (50 ng/ml) for indicated time points and activation of signaling pathways was analyzed by immunoblotting. A representative of two independent experiments is shown.
Figure 2ST2 cells were either left untreated (A) followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) for 30 minutes (B), followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.D-E. Comparison of IL-17-induced transcriptional response in presence or absence of TBK1/IKKε inhibitor. (D) Transcripts that are significantly changed in both conditions. (E) Transcripts that pass significance threshold for induction only in presence of TBK1/IKKε inhibitor. Dashed lines indicate significantly upregulated transcripts (log2 fold change > 1); red lines separates transcripts that are more upregulated upon IL-17 stimulation in the presence of TBK1/IKKε inhibitor as compared to IL-17 alone.F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR. Mean + SEM from five independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting.H. ST2 wild type, TBK1 KO, IKKε KO, or cells lacking both kinases (DKO) were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.I. TBK1/IKKε DKO cells reconstituted with either wild type or kinase dead mutant (K38A) version of both kinases were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.
Figure 3ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ intensities from five independent experiments.ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, statistical significance was determined by two‐tailed Mann-Whitney test.C. ST2 wild type or TBK1/IKKε DKO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 minutes or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1/IKKε DKO cells.D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band.F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.
Figure 4A. ST2 wild type and ACT1-deficient cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.B. ST2 wild type, ACT1 KO or TRAF6 KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting.C. ST2 cells deficient in TRAF6 reconstituted with TRAF6(WT) or enzymatically inactive TRAF6(C70A) were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.D. TRAF6 deficient cells reconstituted with TRAF6(WT), TRAF6(C70A) or empty vector were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated from lysates by anti-Flag immunoprecipitation and analyzed by immunoblotting. Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples, respectively.TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting.TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting (H).ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 or were left unstimulated and IL-17 was added post lysis. results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown (I).
Figure 5ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ values from two independent experiments.ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from two independent experiments is shown.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) and analyzed by immunoblotting.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were tested for activation of signaling pathways (D) and analyzed by immunoblotting.E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.F. ST2 wild type or NEMO KO cells were stimulated with TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.
Figure 6B. Analysis of ACT1 structure using three different algorithms predicting disordered regions. Detected phosphorylation sites are all in the disordered region of the protein.ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.E. ACT1 KO cells expressing either ACT1(WT) or ACT1(9ST mut) were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated via anti-Flag immunoprecipitation and analyzed by immunoblotting.F. ACT1 KO cells were reconstituted with either ACT1(WT) or ACT1(Δ20-380). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.G. Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL-17RC. Data are composite of all 14 MS analyses of IL-17RSC from wild type ST2 cells performed in this study. Mean + SEM is shown.
Figure 1(B) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX. Cut-off for what is regarded as expressed was set to 1.0 NX.
Figure 6(B) ACE2 protein abundance in different human tissues from ProteomicsDB, with the median expression presented in log10(iBAQ).(C) Western blot of ACE2 using five different human tissue lysates. For lung, two different lysates were used: one male (left) and one female (right).
Figure 1(A) Schematic overview of FMR1, FXR1, FXR2, FMR2 and position of the 8 selected single nucleotide polymorphisms (SNPs). Line represents introns, grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons. Gene structure plots generated using FancyGene (Rambaldi Ciccarelli, 2009)(B) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls. (C) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls.
Figure 2Figure 2: SNP overview and unbiased selection according to the standard operating procedure (SOP) developed for phenotype-based genetic association study (PGAS) approaches. (A) Genes from the 'broader fragile X family' and their chromosomal position. (B) SNP numbers available through direct genotyping in the here used semi-custom genotyping array (Affymetrix, Santa Clara, CA, USA). (C) SNPs fulfilling some of the first round of selection criteria ('functional'=SNPs, i.e. located in promoter region, 3'UTR or coding sequence; MAF=MAF≥0.2; LD=SNPs that 'survive' after linkage disequilibrium pruning: r²<0.8). Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS (selection requirements: fulfilled 2 of the above criteria or were functional). Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene. (D) SNPs with a tendency in PGAS (see Fig 3A) at single SNP basis went into the final accumulation model.
Figure 3(A-B) A total of 13 SNPs pre-selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here: (A) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which are eligible for the accumulation model. The genotype associated with the highest average PAUSS (in this example CC) is the 'proautistic genotype' (indicated by the black arrow) and is assigned a score of 1. The heterozygous genotype is assigned a score of 0.5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0. Please note that the difference between genotypes does not have to be statistically significant. (B) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which would not be selected for the accumulation model because of unclear phenotypical/biological relevance.(C-D) The specificity of the association of the 8SNP accumulation model with an autistic phenotype (as determined using PAUSS; compare Fig 4B) is controlled by applying an unrelated (or 'non-sense') phenotype, e.g. delusional-depression: (C) Intercorrelation pattern of single items included in the delusional-depression composite score, used here as example control phenotype. Cronbach's alpha is presented as measure of internal consistency. (D) The delusional-depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.
Figure 4(A) PAUSS (PANSS autism severity score) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals (discovery sample). Cronbach's alpha is presented as measure of internal consistency and also provided for the male replication samples I and II.(B) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.(C) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample; binary logistic regression analysis with non-z-standardized PAUSS as dependent variable; mean±SEM.(D) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients; binary logistic regression analysis; mean±SEM.(E) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients; Mann-Whitney U test; mean±SEM.F) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean±SEM.(G) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample; binary logistic regression analysis; mean±SEM.(H) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population; binary logistic regression analysis; mean±SEM. For all replications p-values for one-sided tests are shown.
Figure 5(A) FXR1 expression in PBMC of individuals carrying the rs2601 GG (low risk; N=16) versus AA genotype (high risk; N=27).(B) PBMC microRNA expression was normalized and data is plotted from all 4 miR-181-5p members (miR-181a, b, c, d-5p).(C) Sequence homology of all 4 human mature miR-181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR-181 family (seed matches identified using Target Scan Human and SFOLD). The red letters specify seed sequences and seed matches, respectively. Chromosome positions for each seed sequence and seed match are denoted (human genome assembly GRCh38/hg38).(D) Denoted are ΔΔG values for binding of each of the miR-181 family members to different 3'UTR positions. Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values. ΔΔG is an energetic score, and the more negative its value, the stronger is the expected binding of the microRNA to the given site
Figure 1(D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.
Figure 2(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).(F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.