EMBO/SourceData huggingface dataset:
figf5(a,b) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype. Where indicated, cells were starved for 5 h before TMRM loading. Where indicated (arrows), 2.5 μg ml−1 oligomycin and 2 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added. Data represent mean ± s.e.m. of seven independent experiments.(c) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments.(d) Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments and are normalized to the initial value.(e,f) Cells of the indicated genotype were starved for the indicated times. Viability was determined by flow cytometry as the percentage of annexin-V- and propidium iodide (PI)-negative events. Data represent mean ± s.e.m. of five independent experiments.(g) MEFs of the indicated genotype were starved for 2.5 h. Where indicated, cells were treated with 20 μM H89. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.(h) Cells of the indicated genotype were starved for the indicated times. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.(i) Drp1−/− MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated. Cell death was determined by flow cytometry as the percentage of YFP- and propidium-iodide-positive events. Data represent mean ± s.e.m. of four independent experiments.(j,k) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.
figf6(a) Blue-native electrophoresis analysis of ATPase dimerization and activity. Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4% digitonin and proteins were separated by blue-native PAGE. ATPase activity was measured in-gel (top) and ATPase levels were measured by immunoblotting for the indicated antibody (bottom). (b,c) Quantitative analysis of the ratio of levels (b) and activity (c) between dimeric and monomeric forms of ATPase, assessed by densitometric analysis of gels from experiments performed as in a. Data represent mean ± s.e.m. of five independent experiments. (d) Experiments were carried out as in a, but with cells of the indicated genotype.
figf7(a) Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h, fixed and processed for electron microscopy. Scale bar, 2 μm. (b) Representative electron micrographs of randomly selected mitochondria from cells of the indicated genotype. Where indicated, cells were starved for 5 h. Scale bars, 0.5 μm. (c) Morphometric analysis of cristae density in cells of the indicated genotype. Experiments were carried out as in a. The number of cristae in 50 randomly selected mitochondria of the indicated genotype was normalized for the calculated surface of the organelle. Data represent mean ± s.e.m. of five independent experiments.
Figure 1(b) Estimation of gene editing at the Trim28 loci using NGS-sequencing of amplicons. Black bars indicate % of frameshift indels. Columns show an average of two biological replicates per guide RNA and error bars show mean ± SD. (c) Western blot confirmed the loss of Trim28 expression upon Trim28-KO in mouse NPCs.  (d) RNA-seq analysis of the expression of TE-families using TEtranscripts (e) The significantly upregulated TE families with a fold change larger than 0.5 upon Trim28-KO in mouse NPCs. The dashed line indicates significance.  (f) CUT&RUN analysis of H3K9me3 in mouse NPCs of all full length MMERVK10Cs (right panel) RNA-seq analysis of the Trim28-KO and control samples, visualising full length MMERVK10C elements. The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.  (g) Example of transcriptional readthrough outside the full length MMERVK10Cs into a nearby gene.
Figure 2 (b) Estimation of gene editing at the Trim28 loci using NGS-sequencing of amplicons from DNA isolated from 50.000 RFP+ nuclei per animal. One animal per group was analyzed. Black bars indicate % of the detected indels that disrupted the frameshift.  Gene editing of the Trim28-loci resulted in a robust loss of Trim28 protein, as evaluated by IHC where the expression of Trim28 in RFP+ cells were quantified and are displayed as mean ± SEM. Approximately 600 RFP+ cells per animal and group was evaluated. Scale bar 30 μm.  Gene editing of the Trim28-loci resulted in a robust loss of Trim28 protein, as evaluated by IHC where the expression of Trim28 in RFP+ cells were quantified and are displayed as mean ± SEM. Approximately 600 RFP+ cells per animal and group was evaluated. Scale bar 30 μm.  (e) RNA-seq analysis of the expression of TE-families using TEtranscripts.  (f) RNA-seq analysis of the Trim28-KO and control samples, visualising full length MMERVK10C elements (left panel) and ChIP-seq analysis of H3K9me3 in adult forebrain neurons (right panel). The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.
Figure 3(b) IHC for Trim28 in the adult cortex revealed that the protein was lost in cells exposed to Cre-activity during brain development (GFP+ cells). Scale bars: low magnification 75 μm, high magnification 20μm. (c) RNA-seq analysis of the expression of TE-families using TEtranscripts (d) Significantly upregulated TE families upon the Trim28-KO, in which the families with the highest fold change are listed.  (e) RNA-seq analysis of full length MMERVK10Cs in the adult tissue. The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.
Figure 4 (e-h) A selection of significant cell type specific changes in gene expression between Emx1-Cre/Trim28-KO animals and controls as revealed by single-nuclei RNA-seq. The black dots represent the mean value value (Wilcoxon rank sum test, (p-adj value < 0.01), n=2.
Figure 5 (b) Mean plots show the changes of TE subfamily expression in each cell cluster upon the Emx1-Cre/Trim28-KO. A differential expression analysis performed with DESeq2 (described in material and methods) showed upregulated TEs in cell types in which Trim28 was deleted (excitatory neurons, astrocytes, oligodendrocytes and oligodendrocyte precursors), (indicated with red dots: p-adj < 0.01, log2FC > 3). The specific upregulated elements and their fold changes are listed in bar graphs under each mean plot. In cell types in which Trim28 was not deleted, TE expression remained unaffected (inhibitory neurons and microglia).
Figure 6 (a) IHC analysis for the microglia marker Iba1 in Emx1-Cre/Trim28-KO animals and controls. Scale bars: Low magnification 75 μm, high magnification 10 μm. (b-c) The morphology of microglia in cortex and striatum (control region) of Emx1-Cre/Trim28-KO animals and controls were quantified by high content screening of Iba1 immunoreactivity, revealing differences in process length, area and number of branch points specifically in cortex, error bars mean ± SEM (unpaired t-test. p-values: 1.8x10-4, 9.6x10-4 and 5.2x10-6, respectively). A large number of photos from three control and two KO animals were analysed  (d) Western blot revealed an increased expression of CD68 in the Emx1-Cre/Trim28-KO animals (n=5 per group), unpaired t-test, **p=0.0036, error bars mean ± SEM. The star indicates an unspecific band.  (e) An increased expression of the ERV derived protein IAP-Gag was detected in the cortex of Emx1-Cre/Trim28-KO animals (n=5 per group), unpaired t-test, ***p=0.0008, error bars mean ± SEM.  (f) Immunohistochemistry for IAP-gag visualized the presence of ERV proteins in the cortex of Emx1-Cre/Trim28-KO animals. The distribution of IAP-gag was heterogenous among Trim28-KO neurons, where it was either accumulated in a low (#) or high (°) number of aggregate-like structures in the cytoplasm, or as weak homogenous staining throughout the cytoplasm (#) or not present at all (*). Scale bars: low magnification 75μm, high magnification 5 μm.
Figure 2(A) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10-wk-old WT and RDEB mice. (B) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age = 5-6. ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.(C) Plot from flow cytometry analysis of CD38+ and Egr2+ macrophages in forepaw skin from 10-wk-old WT and RDEB mice. (D) Bar graph of flow cytometry analyses as in C of inflammatory macrophages (CD38+Egr2-) or tissue-repair macrophages (CD38-Egr2+) macrophages in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age; n = 5-6 (CD38+Egr2-); n = 4-5 (CD38-Egr2+). ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.(F-G) The mean fluorescence intensity (MFI) of MHC II staining on (F) neutrophils (n = 3-4 biological replicates) and (G) macrophages (n = 6-7 biological replicates). Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.
Figure 4(A) Human dermal RDEBF treated daily with 10 μM LY2109761 (LY) (TGF-β Receptor I/II inhibitor‎), 10 μM 1D11 (neutralizing antibody to all TGF-βs) 100 nM Ang-(1-7), or 10 μM losartan (AT1R antagonist). After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins, THBS-1, thrombospondin-1, β-tubulin was used as loading control. (B) Densitometric quantification of blots as in A. The abundance was normalized to β-tubulin and then expressed as % of untreated (-). P values are shown; the data were analyzed with one-way ANOVA with Dunnett's correction (n = 5 biological replicates). ( Data information: P values < 0.05 are considered significant and shown, ns = not significant. individual data points, mean ± S.E.M are shown.(F) Western blots of fibroblast lysates from co-cultures as described in E. The blots were probed with antibodies detecting fibronectin, thrombospondin-1 (THBS-1) and pSMAD-2/3. β-tubulin was used as loading control. (G) Densitometric quantification of western blots of fibroblast cell and matrix lysates as shown in F from multiple independent experiments, n = 4 -5 using cells from 4 donors with RDEB. The abundance was normalized to β-tubulin then and then expressed as % of both untreated. The data were analyzed by one-way ANOVA with Tukey's correction. Data information: P values < 0.05 are considered significant and shown, ns = not significant. For B, D and G individual data points, mean ± S.E.M are shown.
Figure 5(B) Kaplan Meier survival curve of mice treated as in A, P values as indicated, the data were tested with log-rank test - PBS treated vs. 0.1 mg/kg Ang-(1-7) treated and PBS treated vs. 1.0 mg/kg Ang-(1-7) treated; n = 12-15 mice as indicated in the figure.(C) Photos of left and right forepaws of RDEB mice before and after 7 weeks of daily injections with 0.1, 1.0 mg/kg Ang-(1-7) or PBS. (D) Plot of numbers of toes lost during the 7-week observation period, data tested with Kruskal-Wallis test, P values indicated, n= 20-24 paws as indicated in the figure. Median and range are shown. (
Figure 6(A) Haemotoxylin and Eosin (H&E) staining of toes from forepaws from WT and RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Inflammatory cells are elevated in the toes from the PBS and the two Ang-(1-7) treated RDEB mice compared to the WT counterpart. Scale bar = 100 μm. Data information scale bar = 100 μm.(B-D) Flow cytometry analysis of the number of CD45 positive cells (B), percentage of neutrophils (C) and of inflammatory macrophages (CD38+Egr2-) and tissue-repair macrophages (CD38-Egr2+) (D) in forepaw skin of RDEB mice treated as in A. Data information: individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.(F) Staining for IgG (red) in forepaws, nuclei counterstained with DAPI. (G) Quantification of fluorescence staining intensity of samples as in F. Data information: scale bar = 100 μm. , individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.
Figure 7(A) Picrosirius red staining visualized under polarized light and Elastica van Gieson (EvG) staining) of forepaw toes from 12-wk-old WT or RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Note the reduced signal for picrosirus red in RDEB mice treated with 0.1 mg/kg Ang-(1-7) indicating less parallel alignment of and/or thinner collagen fibrils, and additionally the more organized and less disrupted appearance of elastic fibers (black in the EvG staining). (B) Quantification of picrosirius red stained forepaws visualized as in A. A digital threshold was applied to the red channel before quantification. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant; n = 10-15. Individual data points, mean ± S.E.M are shown. ( Data information scale bar = 100 μm(C) Western blotting for the indicated proteins on tissue lysates from forepaws from RDEB mice treated as in A. α-SMA, α-smooth muscle actin; Thbs-1, thrombospondin-1. Data information: β- actin or β-tubulin was used as loading control(D) Dermal β-arrestin-1/2 (red) and NFκB (red) staining in forepaws from WT and RDEB mice treated as in A. Nuclei counterstained with DAPI (blue). Data information scale bar = 20 μm.
Figure 1H&E stained sections of back skin from indicated mice treated with IMQ for 2, 3 or 5 days. Bright-field images, Magnification: 20x, Scale bar: 100 µm.Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were dermal γδ T cells (γδ TCRint+) (F) Graphs show immune cells as % of live, single cells. (n = 10-28 (F), ; ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were Monocytes (CD11b+Ly6Chi) (G), and Neutrophils (CD11b+Ly6G+) (H). Graphs show immune cells as % of live, single cells. n = 10-21 (G), n = 4-25 (H); ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Figure 2H-I. Western blot analysis of Phospho- or total JNK and c-Jun in BMDCs of indicated genotype pre-treated with DMSO (1: 1000) or SP600125 (JNKi, 25 µM) for 1h and stimulated with IMQ (5 µg/mL) for 15 min.Flow cytometry of back skin 12h after intradermal (i.d.) injection of 1 µg rCCL2 and/or IMQ treatment. Analyzed were pDCs (BST-2+B220+CD11cintCD11b-) shown as % of live, single cells. (n = 6; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Tukey multiple comparison test. Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Figure 3Luciferase activity was quantified in lysates of RAW 264.7 cells transfected with pGL3 basic vector harboring wild-type (wt) or mutated (mut1 or 2) IL-23p19 promoter. Transfected cells were pre-treated with JNKi (SP600125, 25 µM, 1h) and stimulated with IMQ (6h). Results are shown as fold change to pGL-3-wt transfected, LAL (Limulus amebocyte lysate) treated RAW264.7 cells. (n = 4; 2 independent experiments).Epidermal thickness of back skin was quantified in H&E stained sections of indicated mice treated with rIL-23 (i.d., 1µg) and/or IMQ for two consecutive days. (n = 4-7; 3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by one-way ANOVA with Tukey multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Figure 4DC subsets (cDC1, cDC2), Macrophages (MP) and Langerhans cells (LC) were sorted from IMQ treated back skin (5h). c-Jun, Ccl2 and Il23p19 mRNA was analyzed by qRT-PCR. Sort strategy is shown in (B). (10 mice were pooled per condition, 1 experiment).
Figure 5Dermal γδ T cells (γδ TCRint+), Monocytes (CD11b+Ly6Chi), and Neutrophils (CD11b+Ly6G+) were analyzed by flow cytometry in mouse ear at the end of treatment (Day 28). Graphs show immune cells as % of live, single cells. (n = 2-8, 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test (N). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Figure 6Immunofluorescence of c-Jun (green), CD1a or CD1c or CD14 (red) and DAPI in psoriatic lesions. Shown is a representative image (Magnification 25x, Scale Bar: 100µm) with an Inset (Magnification 63x, Scale Bar: 20 µm, Deconvoluted). Arrows indicate c-Jun+ DCs. Asterisk highlights a DC with prominent c-Jun expression as shown enlarged in 2 white-framed windows (DC Marker (red) + c-Jun (green, left) or DAPI (blue, right)).D-E. Representative immunofluorescence of c-Jun (green), CD1a (red), CCL2 (D) or Il23p19 (E) (white) and DAPI in psoriatic lesions. Arrows indicate triple-positive cells and an asterisk highlights a representative one that is shown enlarged in a white-framed inlet. Magnification: 40x. Scale bar: 50 µm. (n = 2 patient samples).
Figure 7B-C Human mo-DCs pre-treated with JNKi (SP600125, 25µM) (B) or AP-1 Inhibitor (T-5224, 20 µM) (C) for 1h were stimulated with R848 (10 µg/ml). CCL2 and IL-23 were analyzed by ELISA. (n = 5-11; 2-3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test (B, C, Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.E-F Human moDCs described in (D) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry (E) and CCL2 or IL-23 protein by ELISA (F). (n = 7; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test E and F). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.
Figure 1B. Western blot analysis of hnRNP R in three independent HNRNPR wildtype (+/+), heterozygous (+/-) and homozygous (-/-) HeLa cell lines.C. Confocal microscopy images of nuclei stained with DAPI. Scale bar: 10 µm. D. Quantification of the DAPI area in (C). Data are mean with standard deviation (SD); ***P ≤ 0.001; unpaired two-tailed t-test (n = 3 biological replicates).E. RNA FISH with a Cy3-labeled oligo-dT probe. DAPI was used for nuclear staining. Scale bar: 10 µm. F. Quantification of mean fluorescence intensity of the Cy3 oligo-dT signal in (E). Data are mean with SD; ****P ≤ 0.0001; Mann-Whitney test (n = 129 cells for HNRNPR+/+ and n = 176 cells for HNRNPR-/-).
Figure 2A. Western blot analysis of Ser2-phosphorylated and total RNA pol II, Cyclin T1, CDK9 and GAPDH protein levels.B. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in (A). Data are mean with SD; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates).F. Western blot analysis of subcellular fractions for Calnexin, GAPDH, histone H3, Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins. G. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in subcellular fractions in (F). Data are mean with SD; *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).
Figure 3A. Western blot analysis of the indicated proteins in the input (In) lysates used for co-immunoprecipitation. B. Western blot analysis of Cyclin T1, HEXIM1, CDK9 and hnRNP A1 co-immunoprecipitated by an anti-LARP7 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. C. Western blot analysis of Cyclin T1, HEXIM1, CDK9, hnRNP A1 and LARP7 co-immunoprecipitated by an anti-MePCE antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. D. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with LARP7 in (B). Data are mean with SD; **P ≤ 0.01, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates). E. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with MePCE in (C). Data are mean with SD; *P ≤ 0.05, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates). F. Quantification of hnRNP A1 co-immunoprecipitating with LARP7 and MePCE in (B) and (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-tests (n = 3 biological replicates).G. Western blot analysis of subcellular fractions Calnexin, GAPDH, histone H3, hnRNP R and hnRNP A1. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.H. Western blot analysis of hnRNP R co-immunoprecipitated by an anti-hnRNP A1 antibody from subcellular fractions. Immunoprecipitation with mouse-IgG antibody was used as control.I. Quantification of 7SK RNA in subcellular fractions by qPCR. Data are mean with SD; **P ≤ 0.01, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).J. Quantification of 7SK RNA co-immunoprecipitating with hnRNP A1 from subcellular fractions. Data are mean with SD; *P ≤ 0.05, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).