( a ) Representative electron of of indicated genotype starved where indicated for 5 h , fixed processed for electron microscopy . Scale bar , 2 μm . ( b ) Representative electron of randomly selected mitochondria from of indicated genotype . Where indicated , were starved for 5 h . Scale bars , 0 . 5 μm . ( c ) Morphometric of cristae density in of indicated genotype . Experiments were carried out as in a . number of cristae in 50 randomly selected mitochondria of indicated genotype was normalized for calculated surface of organelle . Data represent mean ± s . e . m . of five independent experiments .

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(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 Trim28 loci using NGS - sequencing of amplicons . Black bars indicate % of frameshift indels . Columns show an average of two biological replicates per guide error bars show mean ± SD .

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(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 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 ) - seq of expression of TE - families using TEtranscripts ( e ) significantly upregulated TE families with a fold change larger than 0 . 5 upon Trim28 - KO in mouse NPCs . dashed indicates significance .

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(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 ) - seq of expression of TE - families using

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


( f ) CUT & RUN of H3K9me3 in mouse NPCs of full MMERVK10Cs ( right panel ) - seq of Trim28 - KO control samples , visualising full MMERVK10C elements . location of full MMERVK10Cs are indicated as a thick black under each histogram .

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(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 full MMERVK10Cs into a nearby gene .

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


( b ) Estimation of gene editing at Trim28 loci using NGS - sequencing of amplicons from isolated from 50 . 000 RFP + nuclei per . One per group was analyzed . Black bars indicate % of detected indels that disrupted frameshift .

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(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 Trim28 - loci resulted in a robust loss of Trim28 , as evaluated by where expression of Trim28 in RFP + were quantified are displayed as mean ± . Approximately 600 RFP + per group was evaluated . Scale bar 30 μm .

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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 ) - seq of expression of TE - families using TEtranscripts .

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


( f ) - seq of Trim28 - KO control samples , visualising full MMERVK10C elements ( left panel ) ChIP - seq of H3K9me3 in adult forebrain neurons ( right panel ) . location of full MMERVK10Cs are indicated as a thick black under each histogram .

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(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 ) for Trim28 in adult cortex revealed that was lost in exposed to Cre - activity during brain development ( GFP + ) . Scale bars : low magnification 75 μm , high magnification 20μm .

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(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 ) - seq of expression of TE - families using TEtranscripts ( d ) Significantly upregulated TE families upon Trim28 - KO , in which families with highest fold change are listed .

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(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 ) - seq of full MMERVK10Cs in adult tissue . location of full MMERVK10Cs are indicated as a thick black under each histogram .

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(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 type specific changes in gene expression between Emx1 - Cre / Trim28 - KO controls as revealed by single - nuclei - seq . black dots represent mean value value ( Wilcoxon rank sum , ( p - adj value < 0 . 01 ) , n = 2 .

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(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 changes of TE subfamily expression in each cluster upon Emx1 - Cre / Trim28 - KO . A differential expression performed with DESeq2 ( described in material methods ) showed upregulated TEs in types in which Trim28 was deleted ( excitatory neurons , astrocytes , oligodendrocytes oligodendrocyte precursors ) , ( indicated with red dots : p - adj < 0 . 01 , log2FC > 3 ) . specific upregulated elements their fold changes are listed in bar graphs under each mean plot . In types in which Trim28 was not deleted , TE expression remained unaffected ( inhibitory neurons microglia ) .

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(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 ) for microglia marker Iba1 in Emx1 - Cre / Trim28 - KO controls . Scale bars : Low magnification 75 μm , high magnification 10 μm . ( b - c ) of microglia in cortex striatum ( control region ) of Emx1 - Cre / Trim28 - KO controls were quantified by high content screening of Iba1 immunoreactivity , revealing differences in process , number of branch points specifically in cortex , error bars mean ± ( unpaired t - . p - values : 1 . 8x10 - 4 , 9 . 6x10 - 4 5 . 2x10 - 6 , respectively ) . A large number of photos from three control two KO were analysed

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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. (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 ) for microglia marker Iba1 in Emx1 - Cre / Trim28 - KO 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 Emx1 - Cre / Trim28 - KO ( n = 5 per group ) , unpaired t - , * * p = 0 . 0036 , error bars mean ± . star indicates an unspecific band .

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(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 ERV derived IAP - Gag was detected in cortex of Emx1 - Cre / Trim28 - KO ( n = 5 per group ) , unpaired t - , * * * p = 0 . 0008 , error bars mean ± .

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(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 presence of ERV in cortex of Emx1 - Cre / Trim28 - KO . 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 cytoplasm , or as weak homogenous throughout cytoplasm ( # ) or not present at ( * ) . Scale bars : low magnification 75μm , high magnification 5 μm .

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(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 of neutrophils in forepaw skin from 10 - wk - old WT RDEB mice . ( B ) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn , 4 - wk - old 10 - wk - old WT RDEB mice . Plotted as fold increase compared to mean of - matched WT paws ; replicates per = 5 - 6 . ( Data information : data points , mean ± S . E . M are shown . data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant .

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(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 of CD38 + Egr2 + macrophages in forepaw skin from 10 - wk - old WT 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 10 - wk - old WT RDEB mice . Plotted as fold increase compared to mean of - matched WT paws ; replicates per ; n = 5 - 6 ( CD38 + Egr2 - ) ; n = 4 - 5 ( CD38 - Egr2 + ) . ( Data information : data points , mean ± S . E . M are shown . data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant .

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(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 ) mean ( MFI ) of MHC II on ( F ) neutrophils ( n = 3 - 4 biological replicates ) ( G ) macrophages ( n = 6 - 7 biological replicates ) . Data information : data points , mean ± S . E . M are shown . data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant .

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(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 ) adult donors ( > 25 years old ) from donors with RDEB with different stages of fibrosis - early ( 0 - 3 years old ) , mild ( 6 - 10 years old ) advanced ( > 15 years old ) - stained for CD68 ( green ) , CD3ε ( green ) , periostin ( green ) tenascin - C ( red ) . Nuclei were counterstained with DAPI ( blue ) . A B , scale bar = 50 μm ; C D , scale bar = 100 μm . bar graphs at right to stained sections , represent for A B of number positive per mm2 analyzed from 3 donors for each group for C D , mean of from 3 - 6 donors per group . data points , mean ± S . E . M are shown . data were analyzed by one - way ANOVA with Tukey ' s correction . P values < 0 . 05 are considered significant .

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(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 TGF - βs ) 100 nM Ang - ( 1 - 7 ) , or 10 μM losartan ( AT1R antagonist ) . After five days of treatment matrix lysates were extracted analyzed by western blotting for indicated , THBS - 1 , thrombospondin - 1 , β - tubulin was used as loading control . ( B ) Densitometric of blots as in A . abundance was normalized to β - tubulin then expressed as % of untreated ( - ) . P values are shown ; data were analyzed with one - way ANOVA with Dunnett ' s correction ( n = 5 biological replicates ) . ( Data information : P values < 0 . 05 are considered significant shown , ns = not significant . data points , mean ± S . E . M are shown .

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(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 . blots were probed with antibodies detecting fibronectin , thrombospondin - 1 ( THBS - 1 ) pSMAD - 2 / 3 . β - tubulin was used as loading control . ( G ) Densitometric of western blots of fibroblast matrix lysates as shown in F from multiple independent experiments , n = 4 - 5 using from 4 donors with RDEB . abundance was normalized to β - tubulin then then expressed as % of both untreated . data were analyzed by one - way ANOVA with Tukey ' s correction . Data information : P values < 0 . 05 are considered significant shown , ns = not significant . For B , D G data points , mean ± S . E . M are shown .

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(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 , data were tested with log - rank - PBS treated vs . 0 . 1 mg / kg Ang - ( 1 - 7 ) treated PBS treated vs . 1 . 0 mg / kg Ang - ( 1 - 7 ) treated ; n = 12 - 15 mice as indicated in figure .

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(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 right forepaws of RDEB mice before after 7 of daily injections with 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS . ( D ) Plot of numbers of toes lost during 7 - week observation period , data tested with Kruskal - Wallis , P values indicated , n = 20 - 24 paws as indicated in figure . Median range are shown . (

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(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 Eosin ( H & E ) of toes from forepaws from WT RDEB mice treated with daily injections of 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS for seven . Inflammatory are elevated in toes from PBS two Ang - ( 1 - 7 ) treated RDEB mice compared to WT counterpart . Scale bar = 100 μm . Data information scale bar = 100 μm .

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(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 of number of CD45 positive ( B ) , of neutrophils ( C ) of inflammatory macrophages ( CD38 + Egr2 - ) tissue - repair macrophages ( CD38 - Egr2 + ) ( D ) in forepaw skin of RDEB mice treated as in A . Data information : data points , mean ± S . E . M are shown . data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant shown ; n = 5 - 14 biological replicates .

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(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 ) for IgG ( red ) in forepaws , nuclei counterstained with DAPI . ( G ) of of samples as in F . Data information : scale bar = 100 μm . , data points , mean ± S . E . M are shown . data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant shown ; n = 5 - 14 biological replicates .

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(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 visualized under polarized Elastica van Gieson ( EvG ) ) 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 . Note reduced signal for picrosirus red in RDEB mice treated with 0 . 1 mg / kg Ang - ( 1 - 7 ) indicating less parallel alignment of / or thinner collagen fibrils , additionally more organized less disrupted appearance of elastic ( black in EvG ) . ( B ) of picrosirius red stained forepaws visualized as in A . A digital threshold was applied to red channel before . data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant ; n = 10 - 15 . data points , mean ± S . E . M are shown . ( Data information scale bar = 100

O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 indicated 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

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(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 ) NFκB ( red ) in forepaws from WT RDEB mice treated as in A . Nuclei counterstained with DAPI ( blue ) . Data information scale bar = 20 μm .

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(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 , Magnification : 20x , Scale bar : 100 µm .

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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 5 days of IMQ treatment . Analyzed were dermal γδ T ( γδ TCRint + ) ( F ) Graphs show immune as % of live , single . ( n = 10 - 28 ( F ) , ; ≥ 2 independent experiments ) . Data information : Data are shown as mean ± . P ‐ values were calculated by Bonferroni multiple comparison 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 .

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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 5 days of IMQ treatment . Analyzed were Monocytes ( CD11b + Ly6Chi ) ( G ) , Neutrophils ( CD11b + Ly6G + ) ( H ) . Graphs show immune as % of live , single . n = 10 - 21 ( G ) , n = 4 - 25 ( H ) ; ≥ 2 independent experiments ) . Data information : Data are shown as mean ± . P ‐ values were calculated by Bonferroni multiple comparison 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 .

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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 of Phospho - or total JNK c - Jun in BMDCs of indicated genotype pre - treated with DMSO ( 1 : 1000 ) or SP600125 ( JNKi , 25 µM ) for 1h stimulated with IMQ ( 5 µg / mL ) for 15 min .

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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 / or IMQ treatment . Analyzed were pDCs ( BST - 2 + B220 + CD11cintCD11b - ) shown as % of live , single . ( n = 6 ; 2 independent experiments ) . Data information : Data are shown as mean ± . P ‐ values were calculated by Tukey multiple comparison . 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 .

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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 transfected with pGL3 basic vector harboring wild - type ( wt ) or mutated ( mut1 or 2 ) IL - 23p19 promoter . Transfected were pre - treated with JNKi ( SP600125 , 25 µM , 1h ) stimulated with IMQ ( 6h ) . Results are shown as fold change to pGL - 3 - wt transfected , LAL ( Limulus amebocyte lysate ) treated RAW264 . 7 . ( n = 4 ; 2 independent experiments ) .

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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 ) / or IMQ for two consecutive days . ( n = 4 - 7 ; 3 independent experiments ) . Data information : Data are shown as mean ± . P ‐ values were calculated by one - way ANOVA with Tukey multiple comparison 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 .

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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 ) Langerhans ( LC ) were sorted from IMQ treated back skin ( 5h ) . c - Jun , Ccl2 Il23p19 mRNA was analyzed by qRT - PCR . Sort strategy is shown in ( B ) . ( 10 mice were pooled per condition , 1 experiment ) .

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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 ( γδ TCRint + ) , Monocytes ( CD11b + Ly6Chi ) , Neutrophils ( CD11b + Ly6G + ) were analyzed by flow cytometry in mouse ear at end of treatment ( Day 28 ) . Graphs show immune as % of live , single . ( n = 2 - 8 , 2 independent experiments ) . Data information : Data are shown as mean ± . P ‐ values were calculated by Bonferroni multiple comparison ( 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 .

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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 ) 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 - framed windows ( DC Marker ( red ) + c - Jun ( green , left ) or DAPI ( blue , right ) ) .

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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 ) ( ) DAPI in psoriatic lesions . Arrows indicate triple - positive an asterisk highlights a representative one that is shown enlarged in a - framed inlet . Magnification : 40x . Scale bar : 50 µm . ( n = 2 samples ) .

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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 IL - 23 were analyzed by ELISA . ( n = 5 - 11 ; 2 - 3 independent experiments ) . Data information : Data are shown as mean ± . P ‐ values were calculated by paired , two - t - ( B , C , Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . See Appendix Table S3 for exact P ‐ values .

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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 of CD80 or CD86 by flow cytometry ( E ) CCL2 or IL - 23 by ELISA ( F ) . ( n = 7 ; 2 independent experiments ) . Data information : Data are shown as mean ± . P ‐ values were calculated by paired , two - t - E F ) . Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . See Appendix Table S3 for exact P ‐ values .

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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 of hnRNP R in three independent HNRNPR wildtype ( + / + ) , heterozygous ( + / - ) homozygous ( - / - ) HeLa lines .

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


C . Confocal microscopy of nuclei stained with DAPI . Scale bar : 10 µm . D . of DAPI in ( C ) . Data are mean with standard deviation ( SD ) ; * * * P ≤ 0 . 001 ; unpaired two - t - ( n = 3 biological replicates ) .

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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 . FISH with a Cy3 - labeled oligo - dT probe . DAPI was used for . Scale bar : 10 µm . F . of mean of Cy3 oligo - dT signal in ( E ) . Data are mean with SD ; * * * * P ≤ 0 . 0001 ; Mann - Whitney ( n = 129 for HNRNPR + / + n = 176 for HNRNPR - / - ) .

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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 of Ser2 - phosphorylated total pol II , Cyclin T1 , CDK9 GAPDH .

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


B . of relative expression of Ser2 - phosphorylated total pol II , Cyclin T1 CDK9 in ( A ) . Data are mean with SD ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; unpaired two - t - ( n = 3 biological replicates ) .

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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 of subcellular fractions for Calnexin , GAPDH , histone H3 , Ser2 - phosphorylated total pol II , Cyclin T1 CDK9 . Cyt , cytosol ; nuc + org , soluble organelles ; chr , - associated . G . of relative expression of Ser2 - phosphorylated total pol II , Cyclin T1 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 ( n = 3 biological replicates ) .

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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 of indicated in input ( In ) lysates used for co - immunoprecipitation . B . Western blot of Cyclin T1 , HEXIM1 , CDK9 hnRNP A1 co - immunoprecipitated by an anti - LARP7 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . C . Western blot of Cyclin T1 , HEXIM1 , CDK9 , hnRNP A1 LARP7 co - immunoprecipitated by an anti - MePCE antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . D . of CDK9 , Cyclin T1 HEXIM1 co - immunoprecipitating with LARP7 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 , n . s . not significant ; unpaired two - t - ( n = 3 biological replicates ) . E . of CDK9 , Cyclin T1 HEXIM1 co - immunoprecipitating with MePCE in ( C ) . Data are mean with SD ; * P ≤ 0 . 05 , n . s . not significant ; unpaired two - t - ( n = 3 biological replicates ) . F . of hnRNP A1 co - immunoprecipitating with LARP7 MePCE in ( B ) ( C ) . Data are mean with SD ; * P ≤ 0 . 05 ; unpaired two - t - ( n = 3 biological replicates ) .

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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 of subcellular fractions Calnexin , GAPDH , histone H3 , hnRNP R hnRNP A1 . Cyt , cytosol ; nuc + org , soluble organelles ; chr , - associated .

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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 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 . of 7SK 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 ( n = 3 biological replicates ) .

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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 . of 7SK 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 ( 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 of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin histone H3 in 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 of hnRNP R , Cyclin T1 , CDK9 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 . of CDK9 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - t - ( 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 of BRD4 ( long 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 . of BRD4 co - immunoprecipitating with CDK9 in ( D ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - t - ( 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 . of histone H3 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - t - ( 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 of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin histone H3 in 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 of hnRNP R , Cyclin T1 , CDK9 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 of BRD4 , hnRNP R , CDK9 α - Tubulin in input lysates used for co - immunoprecipitation .

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


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

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

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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 of Ser2 - phosphorylated total pol II in HNRNPR + / + ( A ) - / - ( B ) treated with DMSO or CHX for indicated durations . GAPDH α - Tubulin served as loading control .

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

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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 of subcellular fractions from treated with DMSO or CHX for Calnexin , GAPDH histone H3 ( D ) Cyt , cytosol ; nuc + org , soluble organelles ; chr , - associated .

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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 of subcellular fractions from treated with DMSO or CHX for Cyclin T1 CDK9 ( E ) . Cyt , cytosol ; nuc + org , soluble organelles ; chr , - associated . F . of relative expression of Cyclin T1 CDK9 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 ( n = 3 biological replicates ) .

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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 of Cyclin T1 in HNRNPR + / + - / - treated with DMSO or MG132 . α - Tubulin served as loading control . H . of relative expression 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 ( n = 3 biological replicates ) .

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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 of Cyclin K , CDK9 α - Tubulin in 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 of Cyclin K co - immunoprecipitated by an anti - CDK9 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

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


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

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

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

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

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. 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 transiently expressing Flag - NEMO , GFP - SHARPIN , HOIL - 1L - HA with Myc - HOIP wildtype ( WT ) , Myc - HOIP K784R or Myc - HOIP C885A . Total 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 .

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

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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 ) CHX ( 1µg / ml ) for 4 hours subjected to 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 .

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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 , Keratin 14 ( KRT14 ) Caspase 3 immunostaining of dorsal skin sections from mice of indicated genotypes at 4 or 8 of . Scale bars : 50 µm .

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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 Caspase 3 ) of mouse dorsal skin sections of indicated genotypes . Scale bars : 50 µm . C

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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


Immunoblotting to examine TNF - induced degradation phosphorylation of IκB - α in immortalized MEFs of indicated genotypes using total extracts of MEFs treated with hTNF ( 20ng / ml ) for indicated times . Representative of three independent experiments . G

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Immunoblotting to examine TNF-induced degradation and phosphorylation of IκB-α in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (20ng/ml) for the indicated times. Representative of three independent experiments. G


Immunoblotting of TNF - induced cleavage of PARP caspase 3 in immortalized MEFs of indicated genotypes using total extracts of MEFs treated with hTNF ( 100ng / ml ) with or without CHX ( 1µg / ml ) for indicated times . Representative of three independent experiments . H

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Immunoblotting of TNF-induced cleavage of PARP and caspase 3 in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) for indicated times. Representative of three independent experiments. H


TNF - dependent induction of Caspase 3 activation in immortalized MEFs of indicated genotype measured by DEVD - AFC . MEFs treated with hTNF ( 100ng / ml ) alone , or with CHX ( 1µg / ml ) for 4 hours ( H ) or 2 hours ( I ) subjected to Caspase 3 activity assays . A representative data of two independent experiments , n = 4 .

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TNF-dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD-AFC. MEFs treated with hTNF (100ng/ml) alone, or with CHX (1µg/ml) for 4 hours (H) or 2 hours (I) subjected to the Caspase 3 activity assays. A representative data of two independent experiments, n=4.


TNF - induced Caspase 8 activity in immortalized MEFs of indicated genotype . Luminol - dependent activity of Caspase 8 in immortalized MEFs treated with hTNF ( 100ng / ml ) with or without CHX ( 1µg / ml ) . A representative data of three independent experiments , n = 4 .

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TNF-induced Caspase 8 activity in immortalized MEFs of the indicated genotype. Luminol-dependent activity of Caspase 8 in immortalized MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) . A representative data of three independent experiments, n=4.


( A ) Representative of GFP ‐ LC3 in HCT116 XIAP WT XIAP KO with stable expression of GFP ‐ LC3 . of LC3 punctate was shown on right . data are represented as means ± s . d . of three independent experiments .

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(A) Representative images of GFP‐LC3 staining in HCT116 XIAP WT and XIAP KO cells with stable expression of GFP‐LC3. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments.


( B ) Endogenous LC3 expression in HCT116XIAP WT XIAP KO was analysed by western blotting with anti ‐ LC3 antibody . data are representative of three biological replicates . of LC3II / LC3I to actin is presented in Supplementary Figure S1A .

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(B) Endogenous LC3 expression in HCT116XIAP WT and XIAP KO cells was analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LC3II/LC3I to actin is presented in Supplementary Figure S1A.


( C ) Lysates from XIAP + / + XIAP − / − mouse embryonic fibroblasts were analysed by western blotting with indicated antibodies . data are representative of two biological replicates .

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(C) Lysates from XIAP+/+ and XIAP−/− mouse embryonic fibroblasts were analysed by western blotting with the indicated antibodies. The data are representative of two biological replicates.


( D ) Autophagic vesicles in HCT116 XIAP WT XIAP KO were evaluated by electron microscopy . are representative of three biological replicates .

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(D) Autophagic vesicles in HCT116 XIAP WT and XIAP KO cells were evaluated by electron microscopy. The images are representative of three biological replicates.


( E ) HCT116 were treated with Embelin as indicated . LC3 ‐ II accumulation was determined by western blot with anti ‐ LC3 antibody . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S1B .

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(E) HCT116 cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1B.


( F ) HCT116 XIAP KO were treated with Embelin as indicated . LC3 ‐ II accumulation was determined by western blot with anti ‐ LC3 antibody . data are representative of three biological replicates .

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(F) HCT116 XIAP KO cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates.


( G ) HCT116 were treated with Leupeptin Embelin in indicated combinations . lysates were analysed by western blotting . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S1D .

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(G) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1D.


( H ) MEF ATG5 WT ATG5 KO were individually transfected with XIAP ‐ specific or control siRNAs . Forty ‐ eight hours after transfection , lysates were analysed by western blotting with indicated antibodies . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S1E .

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(H) MEF ATG5 WT and ATG5 KO cells were individually transfected with XIAP‐specific or control siRNAs. Forty‐eight hours after transfection, cell lysates were analysed by western blotting with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1E.


( I ) HCT116XIAP WT XIAP KO were treated with 20 ‐ μM Z ‐ VAD ‐ FMK or DMSO for 2 h . lysates were then analysed by western blotting . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S1F .

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(I) HCT116XIAP WT and XIAP KO cells were treated with 20 ‐μM Z‐VAD‐FMK or DMSO for 2 h. Cell lysates were then analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1F.


( J ) HCT116 XIAP KO were transfected with either Flag ‐ XIAP ( D148A / W310A ) or control plasmid . Twenty ‐ four hours after transfection , lysates were subjected to western blot with indicated antibodies . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S1G .

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(J) HCT116 XIAP KO cells were transfected with either Flag‐XIAP (D148A/W310A) or control plasmid. Twenty‐four hours after transfection, cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1G.


( A ) A549 IMR90 were transfected with XIAP ‐ specific or control siRNAs . lysates were subjected to western blot with indicated antibodies . data are representative of two biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S2A .

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(A) A549 and IMR90 cells were transfected with XIAP‐specific or control siRNAs. Cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of two biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2A.


( B ) HCT116 expressing XIAP ‐ specific or control siRNAs , HCT116 XIAP WT XIAP KO were individually transfected with Flag ‐ XIAP or control vector . lysates were analysed by western blotting . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S2D .

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(B) HCT116 cells expressing XIAP‐specific or control siRNAs, HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag‐XIAP or control vector. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2D.


( C ) HCT116 XIAP WT XIAP KO were individually transfected with Flag ‐ XIAP or control vector . / fractionation was performed to analyse localization of Mdm2 p53 . PARP GAPDH were used as ( N ) ( C ) fraction markers , respectively . data are representative of three biological replicates .

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(C) HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag‐XIAP or control vector. Cytoplasmic/nuclear fractionation was performed to analyse cellular localization of Mdm2 and p53. PARP and GAPDH were used as nuclear (N) and cytoplasmic (C) fraction markers, respectively. The data are representative of three biological replicates.


( D ) HCT116 p53 + / + p53 − / − were individually transfected with XIAP ‐ specific or control siRNAs . LC3 conversion was detected by western blot . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S2E .

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(D) HCT116 p53+/+ and p53−/− cells were individually transfected with XIAP‐specific or control siRNAs. LC3 conversion was detected by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2E.


( E ) HCT116 p53 + / + p53 − / − were individually transfected with Flag ‐ XIAP or control vector . LC3 conversion was detected by western blot . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S2F .

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(E) HCT116 p53+/+ and p53−/− cells were individually transfected with Flag‐XIAP or control vector. LC3 conversion was detected by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2F.


( F ) HCT116 XIAP WT XIAP KO treated with 10 μM Nutlin3 for 6 h . LC3 conversion was evaluated by western blot . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S2H .

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(F) HCT116 XIAP WT and XIAP KO cells treated with 10 μM Nutlin3 for 6 h. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2H.


( G ) HCT116 XIAP KO were treated with indicated amounts of Nutlin for 8 h . lysates were analysed by western blotting with anti ‐ LC3 antibody . data are representative of three biological replicates .

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(G) HCT116 XIAP KO cells were treated with the indicated amounts of Nutlin for 8 h. Cell lysates were analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates.


( A ) Lysates from HCT116 were immunoprecipitated separately with anti ‐ XIAP antibody an isotype ‐ matched control IgG . Immunoprecipitates input were analysed by western blotting . data are representative of two biological replicates .

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(A) Lysates from HCT116 cells were immunoprecipitated separately with anti‐XIAP antibody and an isotype‐matched control IgG. Immunoprecipitates and input were analysed by western blotting. The data are representative of two biological replicates.


( B ) Recombinant His ‐ XIAP was incubated separately with purified GST GST ‐ Mdm2 fusion on glutathione beads for 4 h followed by western blot using anti ‐ His antibody . GST GST ‐ Mdm2 were analysed by Coomassie blue . data are representative of two biological replicates .

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(B) Recombinant His‐XIAP protein was incubated separately with purified GST and GST‐Mdm2 fusion proteins on glutathione beads for 4 h followed by western blot analysis using anti‐His antibody. GST and GST‐Mdm2 were analysed by Coomassie blue staining. The data are representative of two biological replicates.


( C ) HCT116 were treated with XIAP ‐ specific or control siRNAs . Twenty ‐ four hours later , were cultured in presence of 50 μg ml − 1 cycloheximide for indicated periods of time , subsequently analysed by western blotting . We should mention that amounts of lysates were adjusted to achieve similar expression of Mdm2 at time 0 , while same amounts of lysates were used to examine of XIAP actin . data are representative of three biological replicates . of Mdm2 to actin is presented in Supplementary Figure S3B .

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(C) HCT116 cells were treated with XIAP‐specific or control siRNAs. Twenty‐four hours later, cells were cultured in the presence of 50 μg ml−1 cycloheximide for the indicated periods of time, and subsequently analysed by western blotting. We should mention that amounts of cell lysates were adjusted to achieve similar expression levels of Mdm2 at time 0, while the same amounts of cell lysates were used to examine levels of XIAP and actin. The data are representative of three biological replicates. The ratio of Mdm2 to actin is presented in Supplementary Figure S3B.


( D ) p53 − / − Mdm2 − / − MEF were co ‐ transfected with indicated Mdm2 , XIAP , p53 constructs . lysates were analysed by western blotting with indicated antibodies . We should mention that XIAP H467A expressing plasmid was always used less than XIAP expressing plasmid to ensure expression of XIAP XIAP H467A were similar . data are representative of three biological replicates .

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(D) p53−/−Mdm2−/− MEF cells were co‐transfected with the indicated Mdm2, XIAP, and p53 constructs. Cell lysates were analysed by western blotting with the indicated antibodies. We should mention that XIAP H467A expressing plasmid was always used less than XIAP expressing plasmid to ensure expression levels of XIAP and XIAP H467A were similar. The data are representative of three biological replicates.


( E ) p53 − / − Mdm2 − / − MEF were transfected with indicated plasmids . Twenty ‐ four hours after transfection , were treated with 20 μM MG ‐ 132 for additional 4 h . lysates were denatured before conjugated to His ‐ ubiquitin were pulled down by Ni2 + ‐ NTA beads . bead ‐ bound total lysates ( TCLs ) were analysed by western blot with anti ‐ Mdm2 antibody . data are representative of three biological replicates .

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(E) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins and total cell lysates (TCLs) were analysed by western blot with anti‐Mdm2 antibody. The data are representative of three biological replicates.


( F ) In , 2 μM Mdm2 or its C464A mutant 1 μM XIAP or its H467A mutant were incubated with 100 nM E1 , 2 μM E2 200 μM Ub in a total 20 μlin vitro ubiquitination reaction buffer at 37 ° C for 1 h . reaction mixtures were analysed by western blotting with anti ‐ Mdm2 antibody . data are representative of three biological replicates .

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(F) In all, 2 μM Mdm2 or its C464A mutant and 1 μM XIAP or its H467A mutant proteins were incubated with 100 nM E1, 2 μM E2 and 200 μM Ub in a total 20 μlin vitro ubiquitination reaction buffer at 37°C for 1 h. The reaction mixtures were analysed by western blotting with anti‐Mdm2 antibody. The data are representative of three biological replicates.


( G ) p53 − / − Mdm2 − / − MEF were transfected with indicated plasmids . lysates were analysed by western blotting . data are representative of three biological replicates .

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(G) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Cell lysates were analysed by western blotting. The data are representative of three biological replicates.


( H ) p53 − / − Mdm2 − / − MEF were transfected with indicated plasmids . Twenty ‐ four hours after transfection , were treated with 20 μM MG ‐ 132 for additional 4 h . lysates were denatured before conjugated to His ‐ ubiquitin were pulled down by Ni2 + ‐ NTA beads . bead ‐ bound were analysed by western blotting with anti ‐ p53 antibody . data are representative of three biological replicates .

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(H) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins were analysed by western blotting with anti‐p53 antibody. The data are representative of three biological replicates.


( A ) HCT116 XIAP WT XIAP KO were treated with EBSS for indicated periods of time . GFP ‐ LC3 formation was observed by a microscope . of LC3 punctate was shown on right . data are represented as means ± s . d . of three independent experiments .

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(A) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for the indicated periods of time. GFP‐LC3 puncta formation was observed by a microscope. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments.


( B ) HCT116 XIAP WT XIAP KO were treated with EBSS for 4 h followed by western blot with indicated antibodies . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S6B .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O 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-MEASURED_VAR O O O O O O O O O

(B) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for 4 h followed by western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6B.