D . HoxB8 macrophages were treated as indicated with LPS or LPS + GM - CSF for 12 hours . were lysed NLRP3 was immune precipitated using a magnetic bead coupled antibody . As a negative control , beads without NLRP3 antibody were used . Beads were boiled analysed for recruitment of NLRP3 , caspase - 1 . * indicates chain from antibody . Molecular markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software ( INTAS ) at time of image acquisition . Data information : westerns are representative from 3 biological replicates .

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D. HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF for 12 hours. Cells were lysed and NLRP3 was immune precipitated using a magnetic bead coupled antibody. As a negative control, beads without NLRP3 antibody were used. Beads were boiled and analysed for recruitment of NLRP3, and caspase-1. * indicates light chain from the antibody. Molecular weight markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: westerns are representative images from 3 biological replicates.


E . HoxB8 macrophages were incubated with combinations of LPS , GM - CSF , nigericin + / - Z - VAD . FMK as indicated . were extracted analysed by western blot for caspase - 1 processing . Data information : westerns are representative from 3 biological replicates .

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E. HoxB8 macrophages were incubated with combinations of LPS, GM-CSF, nigericin +/- Z-VAD.FMK as indicated. Proteins were extracted and analysed by western blot for caspase-1 processing. Data information: westerns are representative images from 3 biological replicates.


F . HoxB8 macrophages were seeded in a 96 well microplate co - treated with either 10µM or 50µM z - VAD with LPS + GM - CSF for 16 hours . Then 100nM of Sytox Green was added to . number of sytox green + per image was quantified . Data information : Significance was calculated using one way ANOVA with multiple comparisons . Every dot represents one biological replicate . P values are shown .

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F. HoxB8 macrophages were seeded in a 96 well microplate and co-treated with either 10µM or 50µM z-VAD with LPS+GM-CSF for 16 hours. Then 100nM of Sytox Green was added to the cells. The number of sytox green+ cells per image was quantified. Data information: Significance was calculated using one way ANOVA with multiple comparisons. Every dot represents one biological replicate. P values are shown.


A . HoxB8 macrophages were treated for 16 hours with LPS + / - GM - CSF as indicated . were extracted analysed for of IL - 1β by western blot .

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A. HoxB8 macrophages were treated for 16 hours with LPS +/- GM-CSF as indicated. Proteins were extracted and analysed for levels of IL-1β by western blot.


B . HoxB8 macrophages were treated with LPS or LPS + GM - CSF as indicated for 16 hours . were lysed in buffer containing FLAG tagged K63 TUBE K63 ubiquitylated were precipitated using anti FLAG pull down . Pulled down were analysed for IL - 1β ubiquitin by western blot .

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B. HoxB8 macrophages were treated with LPS or LPS+GM-CSF as indicated for 16 hours. Cells were lysed in buffer containing FLAG tagged K63 TUBE and K63 ubiquitylated proteins were precipitated using anti FLAG pull down. Pulled down proteins were analysed for IL-1β and ubiquitin by western blot.


C . IL - 1β - / - hoxB8 macrophages were infected with lentivirus expressing IL - 1β - FLAG - 6xHis or indicated mutants from IL - 1β promoter . were treated as indicated with LPS or LPS + GM - CSF or LPS + Nigericin of secreted IL - 1β were analysed by ELISA ( top panel ) . GM - CSF treated were also lysed in urea buffer nickel NTA affinity pull down was performed . Nickel NTA pull down was also performed on supernatants ( bottom panel ) . Pulled down form His tag pull down were analysed for presence of IL - 1β by western blot . Molecular markers shown in middle of blot were embedded in image using Chemostar imaging software ( INTAS ) at time of image acquisition . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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C. IL-1β-/- hoxB8 macrophages were infected with lentivirus expressing IL-1β-FLAG-6xHis or the indicated mutants from the IL-1β promoter. Cells were treated as indicated with LPS or LPS+GM-CSF or LPS+Nigericin and levels of secreted IL-1β were analysed by ELISA (top panel). GM-CSF treated cells were also lysed in urea buffer and nickel NTA affinity pull down was performed. Nickel NTA pull down was also performed on supernatants (bottom panel). Pulled down proteins form the His tag pull down were analysed for the presence of IL-1β by western blot. Molecular weight markers shown in middle of blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated . were extracted analysed for IL - 1β by western blot .

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D. HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated. Proteins were extracted and analysed for IL-1β by western blot.


E . Wild type TNF - / - HoxB8 macrophages were treated as indicated with LPS or LPS + GM - CSF . were extracted analysed for IL - 1β by western blot . Black vertical lines indicate that non - relevant lanes were removed during figure preparation but samples were run on same gel .

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E. Wild type and TNF-/- HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF. Proteins were extracted and analysed for IL-1β by western blot. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel.


F . HoxB8 macrophages were treated as in A but with addition of 25µM Punicalagin throughout 16 hour time course . were extracted analysed by western blot ( left panel ) . Supernatants were also analysed by ELISA for secreted IL - 1β ( right panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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F. HoxB8 macrophages were treated as in A but with the addition of 25µM Punicalagin throughout the 16 hour time course. Proteins were extracted and analysed by western blot (left panel). Supernatants were also analysed by ELISA for secreted IL-1β (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


A . Wild type GsdmD - / - HoxB8 macrophages were treated as indicated for 16 hours . Supernatants were analyzed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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A. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


B . HoxB8 macrophages were seeded in an 8 - well Ibidi chamber treated for 14 hours with LPS GM - CSF . were fixed stained for GasderminD ( green ) , IL - 1β ( red ) DAPI ( blue ) . were taken at Zeiss LSM880 Confocal microscope Data information : shown . Microscopy pictures are representative from 3 biological replicates . Scale bars in microscopy represent 10µM .

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B. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated for 14 hours with LPS and GM-CSF. Cells were fixed and stained for GasderminD (green), IL-1β (red) and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope Data information: shown. Microscopy pictures are representative images from 3 biological replicates. Scale bars in microscopy images represent 10µM.


C . HoxB8 macrophages were co - stimulated with three different PEG 3mM for 16 hours as indicated . Supernatants were analyzed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages were co-stimulated with three different PEG 3mM for 16 hours as indicated. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 macrophages were seeded in 96 well microplate treated as indicated . Before treatment , sytox green was added were imaged in an incucyte incubator microscope every hour for 16 hours . Shown is sytox green signal normalized to total . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For panels , error bars are . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages were seeded in 96 well microplate and treated as indicated. Before treatment, sytox green was added and cells were imaged in an incucyte incubator microscope every hour for 16 hours. Shown is the sytox green signal normalized to the total cell area. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


E . Wild type GsdmD - / - HoxB8 macrophages were treated as indicated for 16 hours . were extracted analysed for IL - 1β ubiquitination by western blot . Data information : westerns are representative from 3 biological replicates .

O O O O O O O O O O O O O O O O O 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. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Proteins were extracted and analysed for IL-1β ubiquitination by western blot. Data information: westerns are representative images from 3 biological replicates.


( a ) Representative confocal of mitochondrial in MEFs of indicated genotype 24 h after transfection with mtYFP . Where indicated , were starved for 2 . 5 h . Scale bar , 20 μm . ( b ) Morphometric of mitochondrial shape . Experiments were carried out as in a . Data represent mean ± s . e . m . of three independent experiments ( n = 100 per condition in each experiment ) .

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(a) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP. Where indicated, cells were starved for 2.5 h. Scale bar, 20 μm. (b) Morphometric analysis of mitochondrial shape. Experiments were carried out as in a. Data represent mean ± s.e.m. of three independent experiments (n=100 cells per condition in each experiment).


( c ) Top left , 48 h after transfection with indicated siRNA , MEFs were lysed 25 μg samples of were separated by DS - polyacrylamide gel electrophoresis ( PAGE ) immunoblotted with indicated antibodies . Top right bottom , representative show mitochondrial of MEFs transfected with indicated siRNA after 24 h with mtYFP . After a further 24 h , confocal were acquired . Scale bar , 20 μm . Uncropped of blots are shown in Supplementary Fig . S8 . ( d ) Morphometric of mitochondrial shape . Experiments were carried out as in c . Data represent mean ± s . e . m . of five independent experiments ( n = 100 per condition in each experiment ) .

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(c) Top left, 48 h after transfection with the indicated siRNA, MEFs were lysed and 25 μg samples of proteins were separated by DS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. Top right and bottom, representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP. After a further 24 h, confocal micrographs were acquired. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S8. (d) Morphometric analysis of mitochondrial shape. Experiments were carried out as in c. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition in each experiment).


( e ) Representative of mitochondrial fusion over time ( indicated in min ) . MEFs were co - transfected with mtPAGFP mtRFP after 24 h , mtPAGFP was photoactivated in a region of interest were imaged by real - time confocal microscopy . Where indicated , MEFs were starved for 2 . 5 h . Scale bar , 20 μm . See also Supplementary Movies S1 S2 . ( f , g ) of mitochondrial fusion in MEFs of indicated genotype . Experiments were carried out as in e . Data represent mean ± s . e . m . of four independent experiments .

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(e) Representative images of mitochondrial fusion over time (indicated in min). MEFs were co-transfected with mtPAGFP and mtRFP and after 24 h, mtPAGFP was photoactivated in a region of interest and cells were imaged by real-time confocal microscopy. Where indicated, MEFs were starved for 2.5 h. Scale bar, 20 μm. See also Supplementary Movies S1 and S2. (f,g) Quantification of mitochondrial fusion in MEFs of the indicated genotype. Experiments were carried out as in e. Data represent mean ± s.e.m. of four independent experiments.


( h ) Representative electron of muscle ( longitudinal sections ; four top ) liver ( four bottom ) from CD1 mice . Where indicated , mice were fasted for 12 h . outlined regions of from muscle samples are magnified × 7 in below . Scale bars , 2 μm .

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(h) Representative electron micrographs of muscle (longitudinal sections; four top micrographs) and liver (four bottom micrographs) from CD1 mice. Where indicated, mice were fasted for 12 h. The outlined regions of micrographs from muscle samples are magnified ×7 in the images below. Scale bars, 2 μm.


( a - d ) of mitochondria - shaping during starvation . samples ( 20 μg ) from MEFs of indicated genotype were separated by SDS - PAGE immunoblotted with indicated antibodies . were starved for indicated times .

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(a-d) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times.


( e ) Association of DRP1 with mitochondria during starvation . Mitochondria were isolated from MEFs starved for indicated times 25 μg samples of were separated by SDS - PAGE immunoblotted with indicated antibodies . ( f ) of Ser 637 phosphorylation of DRP1 during starvation . Equal amounts of lysates from wild - type MEFs starved for indicated times were immunoprecipitated with indicated antibody immunoprecipitated were separated by SDS - PAGE immunoblotted with indicated antibodies . ( g ) Quantitative of Ser 637 phosphorylation of DRP1 during starvation . Experiments were carried out as in f . Data are normalized to total of DRP1 represent mean ± s . e . m . of three independent experiments .

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(e) Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (f) Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (g) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in f. Data are normalized to total levels of DRP1 and represent the mean ± s.e.m. of three independent experiments.


( h ) MEFs were treated for indicated times with 100 nM rapamycin or with 25 μM forskolin ( FRSK ) for 0 . 5 h , lysed equal amounts ( 50 μg ) of were separated by SDS - PAGE immunoblotted using indicated antibodies .

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(h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies.


( i ) HeLa were transfected for 2 days with indicated siRNA or treated with 25 μM forskolin for 0 . 5 h lysed . Equal amounts ( 50 μg ) of were separated by SDS - PAGE immunoblotted using indicated antibodies . Uncropped of blots in this figure are shown in Supplementary Fig . S8 .

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(i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


( a ) Pseudocolour - coded of EPACI - camps FRET from real - time imaging of wild - type MEFs transfected with EPACI - camps . Where indicated , were perfused with starvation solution for indicated times ( in min ) or with 25 μM forskolin ( FRSK ) . Scale bar , 20 μm . Colour scale indicates F1 / F0 . See also Supplementary Movie S3 . ( b ) Quantitative of CFP / YFP FRET . Experiments were carried out as in a . Where indicated , were perfused with starvation solution or with 25 μM forskolin . Data represent mean ± s . e . m . of 13 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 O O O O 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 O O 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 O O O O O O O O O O O O O O O O

(a) Pseudocolour-coded images of EPACI-camps FRET from real-time imaging of wild-type MEFs transfected with EPACI-camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments.


( c ) Lysate samples ( 50 μg ) from MEFs of indicated genotypes were analysed by SDS - PAGE immunoblotting using indicated antibodies . Where indicated , MEFs were starved , or treated with 25 μM forskolin . Where indicated , 20 μM H89 was added during starvation . Uncropped of blots in this figure are shown in Supplementary Fig . S8 .

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(c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


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

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(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 of mitochondrial of wild - type MEFs co - transfected with mtRFP indicated plasmids . At 24 h after transfection , where indicated were starved for 2 . 5 h imaged . Where indicated , 20 μM H89 was present during starvation . Scale bar , 20 μm . ( g ) Morphometric of mitochondrial shape . Experiments were carried out as in f . Data represent mean ± s . e . m . of five independent experiments ( n = 50 per condition ) .

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

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

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

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(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 of TMRM changes over mitochondrial regions in MEFs of indicated genotype . Where indicated , were starved for 5 h before TMRM loading . Where indicated ( arrows ) , 2 . 5 μg ml − 1 oligomycin 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 O O O O O O O O O O O O O O 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 O O O O O O O O O O B-CONTROLLED_VAR 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 ATP were measured in of indicated genotype starved for indicated times . Data represent mean ± s . e . m . of five independent experiments .

O O O O B-MEASURED_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

(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 of indicated genotype starved for indicated times . Data represent mean ± s . e . m . of five independent experiments are normalized to initial value .

O O O O B-MEASURED_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

(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 ) of indicated genotype were starved for indicated times . Viability was determined by flow cytometry as of annexin - V - propidium iodide ( PI ) - negative events . 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 O O O O O O O O O O O

(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 indicated genotype were starved for 2 . 5 h . Where indicated , were treated with 20 μM H89 . 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 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

(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 ) of indicated genotype were starved for 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

(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 indicated plasmids after 24 h starved for 5 h where indicated . was determined by flow cytometry as of YFP - propidium - iodide - positive events . Data represent mean ± s . e . m . of four 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 O O O O O O O O O O O O O O O O O

(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 indicated genotype were starved for 5 h in 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 .

O O O 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 O O O 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,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 of ATPase dimerization activity . of indicated genotype were treated as indicated 500 μg of total extract was solubilized with 4 % digitonin were separated by blue - native PAGE . ATPase activity was measured in - gel ( top ) ATPase were measured by immunoblotting for indicated antibody ( bottom ) . ( b , c ) Quantitative of of ( b ) activity ( c ) between dimeric monomeric forms of ATPase , assessed by densitometric 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 of indicated genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O 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 loss of Trim28 expression upon Trim28 - KO in mouse NPCs .

(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 .

O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

(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 .

O O O O O O O O O O O O O O O O O O O O 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 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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O 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) 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 .

O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O 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.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ± .

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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 .

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 ) .

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 ) .

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 .

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