( d ) Representative images of the effect of H89 on mitochondrial morphology on starvation . Wild - type and Mfn2 − / − MEFs were transfected with mtYFP , and after 24 h confocal micrographs were acquired . Where indicated , cells were starved for 2 . 5 h and 20 μM H89 was added . Scale bar , 20 μm . ( e ) Morphometric analysis . Experiments were carried out as in d . Data represent mean ± s . e . m . of five independent experiments ( n = 100 cells per condition ) .

O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Representative images of the effect of H89 on mitochondrial morphology on starvation. Wild-type and Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. (e) Morphometric analysis. Experiments were carried out as in d. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition).


( f ) Starvation - induced mitochondrial elongation depends on Ser 637 of DRP1 . Representative confocal micrographs of mitochondrial morphology of wild - type MEFs co - transfected with mtRFP and the indicated plasmids . At 24 h after transfection , where indicated cells were starved for 2 . 5 h and imaged . Where indicated , 20 μM H89 was present during starvation . Scale bar , 20 μm . ( g ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in f . Data represent mean ± s . e . m . of five independent experiments ( n = 50 cells per condition ) .

O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-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 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 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 O O O O O O

(f) Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (g) Morphometric analysis of mitochondrial shape. Experiments were carried out as in f. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).


( h ) Representative confocal micrographs of mitochondrial morphology of Drp1 − / − MEFs co - transfected with mtRFP and the indicated plasmids . At 24 h after transfection , where indicated cells were starved for 2 . 5 h and imaged . Where indicated , 20 μM H89 was present during starvation . Scale bar , 20 μm . ( i ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in h . Data represent mean ± s . e . m . of five independent experiments ( n = 50 cells per condition ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O B-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 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 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 O O O O O O

(h) Representative confocal micrographs of mitochondrial morphology of Drp1−/− MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (i) Morphometric analysis of mitochondrial shape. Experiments were carried out as in h. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).


( a - c ) MEFs of the indicated genotype were treated as indicated , counted and 2 . 7 × 105 cells were lysed . Lysates were separated by SDS - PAGE and immunoblotted using the indicated antibodies . ( d ) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype . One representative experiment of five independent repetitions performed as in a - c is shown .

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

(a-c) MEFs of the indicated genotype were treated as indicated, counted and 2.7×105 cells were lysed. Lysates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (d) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype. One representative experiment of five independent repetitions performed as in a-c is shown.


( e ) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0 . 5 μM wortmannin ( Wortm . ) . Lysates from 2 . 7 × 105 cells were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

O O O B-CELL_LINE 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 O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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

(e) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0.5 μM wortmannin (Wortm.). Lysates from 2.7×105 cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


( 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 .

O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-CELL_LINE O O 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 B-SMALL_MOLECULE 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 O O O O O

(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 .

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 O O O O O O

(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 .

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(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 .

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(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 .

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(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 .

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

(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 .

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(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 .

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(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.


( 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 .

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

(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.


( 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 .

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 O O O O O O O O O O O O O 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 O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O 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 O O

(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.


( 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 .

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

(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 .

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(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 .

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

(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.


( d ) RNA - seq analysis of the expression of TE - families using

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O

(d) RNA-seq analysis of the expression of TE-families using TEtranscripts


( 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 .

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

(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 .

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

(g) Example of transcriptional readthrough outside the full length MMERVK10Cs into a nearby gene.


( 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 .

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

(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 .

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

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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O

(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 .

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

(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.


( 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 .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY 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

(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 .

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

(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.


( 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 .

O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-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

(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.


( 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 ) .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O O B-CELL_TYPE O B-CELL_TYPE O O

(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).


( 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

O O O B-EXP_ASSAY O O O B-CELL_TYPE 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 B-EXP_ASSAY O B-CELL_TYPE O B-TISSUE O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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


( 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 .

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(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.


( 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 .

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

(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 .

O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-TISSUE 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

(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 .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O 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-SUBCELLULAR O O O O O B-EXP_ASSAY O 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

(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.


( 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


( A - D ) Skin from healthy young ( 3 - 8 years old ) and adult donors ( > 25 years old ) and from donors with RDEB with different stages of fibrosis - early ( 0 - 3 years old ) , mild ( 6 - 10 years old ) and advanced ( > 15 years old ) - stained for CD68 ( green ) , CD3ε ( green ) , periostin ( green ) and tenascin - C ( red ) . Nuclei were counterstained with DAPI ( blue ) . A and B , scale bar = 50 μm ; C and D , scale bar = 100 μm . The bar graphs at the right to the stained sections , represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D , the mean intensity of staining from 3 - 6 donors per group . 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 .

O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O B-DISEASE O O O 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 O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY 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 B-EXP_ASSAY 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 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

(A-D) Skin from healthy young (3-8 years old) and adult donors ( > 25 years old) and from donors with RDEB with different stages of fibrosis - early (0-3 years old), mild (6-10 years old) and advanced ( > 15 years old) - stained for CD68 (green), CD3ε (green), periostin (green) and tenascin-C (red). Nuclei were counterstained with DAPI (blue). A and B, scale bar = 50 μm; C and D, scale bar = 100 μm. The bar graphs at the right to the stained sections, represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D, the mean intensity of staining from 3-6 donors per group. 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.


( 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 .

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

(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 .

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

(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.


( 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 .

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

(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 . (

O O O O O O O O B-TISSUE O B-DISEASE B-ORGANISM 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 I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O

(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. (


( 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 .

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

(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 .

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

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-TISSUE O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE 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 O O O O 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) 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.


( 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

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-DISEASE B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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

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

(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 .

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

(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.


H & 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 .

O O O B-EXP_ASSAY O O O B-TISSUE O O B-ORGANISM O O B-SMALL_MOLECULE O O O 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

H&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 .

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

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 .

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

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.


H - 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 .

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

H-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 .

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

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.


Luciferase 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 ) .

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

Luciferase 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 .

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

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.


DC 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 ) .

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

DC 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).


Dermal 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 .

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

Dermal 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.


Immunofluorescence 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 ) ) .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-DISEASE I-DISEASE O O O O O O O O O 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 I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O

Immunofluorescence 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 ) .

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

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).


B - 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 .

O O O B-ORGANISM B-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

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

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.


B . Western blot analysis of hnRNP R in three independent HNRNPR wildtype ( + / + ) , heterozygous ( + / - ) and homozygous ( - / - ) HeLa cell lines .

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

B. 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE 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 O O 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. 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 - / - ) .

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

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-/-).


A . Western blot analysis of Ser2 - phosphorylated and total RNA pol II , Cyclin T1 , CDK9 and GAPDH protein levels .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O

A. 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 ) .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-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 O O O O O O O O O O

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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-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 O O O O O O O O O O O O O O O O

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).


A . 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 ) .

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

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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 .

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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 ) .

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

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 ) .

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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).


A . Western blot analysis of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin and histone H3 in the input lysates used for co - immunoprecipitation .

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A. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation.


B . Western blot analysis of hnRNP R , Cyclin T1 , CDK9 and histone H3 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

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B. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


C . Quantification of CDK9 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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C. Quantification of CDK9 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


D . Western blot analysis of BRD4 ( long and short exposure ) co - immunoprecipitated by an anti - CDK9 antibody . Immunoprecipitation with mouse - IgG antibody was used as control .

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D. Western blot analysis of BRD4 (long and short exposure) co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with mouse-IgG antibody was used as control.


E . Quantification of BRD4 co - immunoprecipitating with CDK9 in ( D ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

E. Quantification of BRD4 co-immunoprecipitating with CDK9 in (D). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


F . Quantification of histone H3 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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F. Quantification of histone H3 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


G . Western blot analysis of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin and histone H3 in the input lysates used for co - immunoprecipitation . Lysates were pre - treated with RNase as indicated .

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G. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation. Lysates were pre-treated with RNase as indicated.


H . Western blot analysis of hnRNP R , Cyclin T1 , CDK9 and histone H3 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

H. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


I . Western blot analysis of BRD4 , hnRNP R , CDK9 and α - Tubulin in the input lysates used for co - immunoprecipitation .

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

I. Western blot analysis of BRD4, hnRNP R, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.


J . Western blot analysis of hnRNP R and CDK9 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . K . Quantification of CDK9 co - immunoprecipitating with BRD4 in ( J ) . Data are mean with SD ; * * * * P ≤ 0 . 0001 ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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

J. Western blot analysis of hnRNP R and CDK9 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. K. Quantification of CDK9 co-immunoprecipitating with BRD4 in (J). Data are mean with SD; ****P ≤ 0.0001; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


L . Western blot analysis of phospho - CDK9 ( Thr186 ) co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . M . Quantification of phospho - CDK9 ( Thr186 ) co - immunoprecipitating with BRD4 in ( L ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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

L. Western blot analysis of phospho-CDK9(Thr186) co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. M. Quantification of phospho-CDK9(Thr186) co-immunoprecipitating with BRD4 in (L). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


A , B . Western blot analysis of Ser2 - phosphorylated and total RNA pol II levels in HNRNPR + / + ( A ) and - / - ( B ) cells treated with DMSO or CHX for the indicated durations . GAPDH and α - Tubulin served as loading control .

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

A, B. Western blot analysis of Ser2-phosphorylated and total RNA pol II levels in HNRNPR+/+ (A) and -/- (B) cells treated with DMSO or CHX for the indicated durations. GAPDH and α-Tubulin served as loading control.


C . Quantification of relative expression levels of Ser2 - phosphorylated and total RNA pol II in ( A , B ) . Data are mean with SD ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ; unpaired two - tailed t - tests ( n = 4 biological replicates ) .

O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-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 O 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. Quantification of relative expression levels of Ser2-phosphorylated and total RNA pol II in (A, B). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; unpaired two-tailed t-tests (n = 4 biological replicates).


D , Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Calnexin , GAPDH and histone H3 ( D ) Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins .

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

D, Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Calnexin, GAPDH and histone H3 (D) Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.


E . Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Cyclin T1 and CDK9 ( E ) . Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins . F . Quantification of relative expression of Cyclin T1 and CDK9 protein levels in subcellular fractions in ( E ) . Data are mean with SD ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; one - way ANOVA with Tukey ' s multiple comparisons test ( n = 3 biological replicates ) .

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

E. Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Cyclin T1 and CDK9 (E). Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins. F. Quantification of relative expression of Cyclin T1 and CDK9 protein levels in subcellular fractions in (E). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; one-way ANOVA with Tukey's multiple comparisons test (n = 3 biological replicates).


G . Western blot analysis of Cyclin T1 levels in HNRNPR + / + and - / - cells treated with DMSO or MG132 . α - Tubulin served as loading control . H . Quantification of relative expression levels of Cyclin T1 in ( G ) . Data are mean with SD ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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

G. Western blot analysis of Cyclin T1 levels in HNRNPR+/+ and -/- cells treated with DMSO or MG132. α-Tubulin served as loading control. H. Quantification of relative expression levels of Cyclin T1 in (G). Data are mean with SD; ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


B . Western blot analysis of Cyclin K , CDK9 and α - Tubulin in the input lysates used for co - immunoprecipitation .

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B. Western blot analysis of Cyclin K, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.


C . Western blot analysis of Cyclin K co - immunoprecipitated by an anti - CDK9 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

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

C. Western blot analysis of Cyclin K co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


D . Quantification of Cyclin K co - immunoprecipitating with CDK9 in ( C ) . Data are mean with SD ; * P ≤ 0 . 05 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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

D. Quantification of Cyclin K co-immunoprecipitating with CDK9 in (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-test (n = 3 biological replicates).


Effects of ubiquitin surface mutants based on the UbcH7 - Ub : R0RBR : pUb model for the ubiquitin loading and off - loading potential of pParkinCH ( pParkinC431S , H433A ) . Coomassie stained gels ( top ) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species . Anti - HA blots ( bottom ) depict the ubiquitination of HA - tagged Miro1181 - 579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species . The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin .

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

Effects of ubiquitin surface mutants based on the UbcH7-Ub:R0RBR:pUb model for the ubiquitin loading and off-loading potential of pParkinCH (pParkinC431S,H433A). Coomassie stained gels (top) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species. Anti-HA blots (bottom) depict the ubiquitination of HA-tagged Miro1181-579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species. The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin.


Auto - ubiquitination assay for parkin using ARJP and non - ARJP substitutions in parkin observed near the interface with UbcH7 - Ub . Assays were done in the absence and presence of pUb . Experiments with PINK1 were done by treating parkin : pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination . Assays were monitored using a DyLight - labelled Ub protein and measuring fluorescence at 800 nm .

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Auto-ubiquitination assay for parkin using ARJP and non-ARJP substitutions in parkin observed near the interface with UbcH7-Ub. Assays were done in the absence and presence of pUb. Experiments with PINK1 were done by treating parkin:pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination. Assays were monitored using a DyLight-labelled Ub protein and measuring fluorescence at 800 nm.


Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe . The different stages of activation using parkin , parkin : pUb , pParkin : pUb and pParkin : pUb in combination with an isopeptide linked UbcH7 - Ub conjugate are indicated above each gel . Following addition of UbVS , samples were taken at the times indicated ( 0 - 10 minutes ) and visualized by SDS - PAGE . Relative percentages of pParkin and the pParkin - Ub adduct as a function of time . Intensity percentages were calculated as a function of total intensity of pParkin - Ub , parkin and UbVS / pUb bands . Error bars represent standard deviation from the average for duplicate measurements . Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe . The different stages of activation using R0RBR , R0RBR : pUb , R0RBR : pUb : pUbl and R0RBR : pUb : pUbl in combination with an isopeptide linked UbcH7 - Ub conjugate are indicated above each gel . Following addition of UbVS , samples were taken at the times indicated ( 0 - 60 minutes ) and visualized by SDS - PAGE Relative percentages of R0RBR and the R0RBR - Ub adduct as a function of time . Intensity percentages were calculated as a function of total intensity of the R0RBR - Ub , R0RBR and UbVS / pUb / pUbl bands .

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

Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe. The different stages of activation using parkin, parkin:pUb, pParkin:pUb and pParkin:pUb in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-10 minutes) and visualized by SDS-PAGE.   Relative percentages of pParkin and the pParkin-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of pParkin -Ub, parkin and UbVS/pUb bands. Error bars represent standard deviation from the average for duplicate measurements.  Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe. The different stages of activation using R0RBR, R0RBR:pUb, R0RBR:pUb:pUbl and R0RBR:pUb:pUbl in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-60 minutes) and visualized by SDS-PAGE   Relative percentages of R0RBR and the R0RBR-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of the R0RBR-Ub, R0RBR and UbVS/pUb/pUbl bands.


. Co - immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL - 1L using total cell extracts of HEK293T cells transiently expressing Myc - HOIP wildtype ( WT ) , or Myc - HOIP - K784R with HOIL - 1L - HA and Flag - SHARPIN . Anti - Vinculin antibody used to monitor protein loading . Representative data shown from three independent experiments .

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

. Co-immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL-1L using total cell extracts of HEK293T cells transiently expressing Myc-HOIP wildtype (WT), or Myc-HOIP-K784R with HOIL-1L-HA and Flag-SHARPIN. Anti-Vinculin antibody used to monitor protein loading. Representative data shown from three independent experiments.


. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag - NEMO , GFP - SHARPIN , and HOIL - 1L - HA with Myc - HOIP wildtype ( WT ) , Myc - HOIP K784R or Myc - HOIP C885A . Total cell lysates in denaturing conditions subjected to SDS - PAGE followed up by immunoblotting using antibodies as indicated . Anti - Vinculin antibody to monitor loading . Representative data from three independent experiments .

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

. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag-NEMO, GFP-SHARPIN, and HOIL-1L-HA with Myc-HOIP wildtype (WT), Myc-HOIP K784R or Myc-HOIP C885A. Total cell lysates in denaturing conditions subjected to SDS-PAGE followed up by immunoblotting using antibodies as indicated. Anti-Vinculin antibody to monitor loading. Representative data from three independent experiments.


Immunoblotting to detect TNF - induced degradation and phosphorylation of IκB - α , or phosphorylation of IKK in immortalized Hoip + / + and HoipK778R / K778R MEFs treated with human TNF ( 20ng / ml ) for the indicated times . Immunoblots of anti - Vinculin antibody and anti - α - Tubulin antibody shown for monitoring loading amount . Representative data from three independent

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

Immunoblotting to detect TNF-induced degradation and phosphorylation of IκB-α, or phosphorylation of IKK in immortalized Hoip+/+ and HoipK778R/K778R MEFs treated with human TNF (20ng/ml) for the indicated times. Immunoblots of anti-Vinculin antibody and anti-α-Tubulin antibody shown for monitoring loading amount. Representative data from three independent experiments


TNF - dependent induction of Caspase 3 activation in immortalized Hoip + / + or HoipK778R / K778R MEFs measured by using DEVD - AFC . MEFs treated with hTNF ( 100ng / ml ) and CHX ( 1µg / ml ) for 4 hours subjected to the Caspase 3 activity assays .

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TNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours subjected to the Caspase 3 activity assays.


TNF - induced Caspase 8 activity in Hoip + / + or HoipK778R / K778R immortalized MEFs treated with hTNF ( 100ng / ml ) with or without Cycloheximide ( CHX ) ( 1µg / ml ) or z - VAD ( 20µM ) . Representative data from three independent experiments , n = 4 .

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

TNF-induced Caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF (100ng/ml) with or without Cycloheximide (CHX) (1µg/ml) or z-VAD (20µM). Representative data from three independent experiments, n=4.


H & E staining , Keratin 14 ( KRT14 ) and Cleaved Caspase 3 immunostaining of dorsal skin sections from mice of the indicated genotypes at 4 or 8 weeks of age . Scale bars : 50 µm .

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

H&E staining, Keratin 14 (KRT14) and Cleaved Caspase 3 immunostaining of dorsal skin sections from mice of the indicated genotypes at 4 or 8 weeks of age. Scale bars: 50 µm.


Immunostaining ( H & E , Keratin 14 ( KRT14 ) , Ly6G , F4 / 80 and Cleaved Caspase 3 ) of mouse dorsal skin sections of the indicated genotypes . Scale bars : 50 µm . C

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

Immunostaining (H&E, Keratin 14 (KRT14), Ly6G, F4/80 and Cleaved Caspase 3) of mouse dorsal skin sections of the indicated genotypes. Scale bars: 50 µm. C