( B ) Three fractions representing the 150S and 75S peaks were pooled and run on SDS - PAGE as in Figure 6 . The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units . The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay . * p < 0 . 05 .

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(B) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE as in Figure 6. The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units. The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay. * p<0.05.


C ) Immunofluorescence image of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against CENP - T . Images in C maximum intensity projections taken across the depth of the EGFP foci .

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C) Immunofluorescence image of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against CENP-T. Images in C maximum intensity projections taken across the depth of the EGFP foci.


E ) Quantification of CENP - T signal intensity at MUC4 ectopic foci vs . endogenous centromeres , normalised to CENP - T signal intensity at endogenous centromeres ( = 1 , red line ) . LinesLines = Mean ± SD , 3 experiments , each with ≥ 10 metaphases analysed per condition . Each point = 1 focus or centromere . ns = p > 0 . 05 , * * * = p < 0 . 01 , * * * * = p < 0 . 001 ( Kruskal Wallis test with Dunn ' s multiple comparison correction ) .

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E) Quantification of CENP-T signal intensity at MUC4 ectopic foci vs. endogenous centromeres, normalised to CENP-T signal intensity at endogenous centromeres (=1, red line). LinesLines = Mean ± SD, 3 experiments, each with ≥ 10 metaphases analysed per condition. Each point = 1 focus or centromere. ns = p>0.05, *** = p<0.01, **** = p<0.001 (Kruskal Wallis test with Dunn's multiple comparison correction).


H ) Quantification of KNL ‑ 1 signal intensity at MUC4 foci , normalised to KNL ‑ 1 signal intensity at centromeres ( = 1 , red line ) . LinesLines = Mean ± SD , 3 experiments , each with ≥ 10 metaphases analysed per condition . Each point = 1 focus or centromere . ns = p > 0 . 05 , * * * = p < 0 . 01 , * * * * = p < 0 . 001 ( Kruskal Wallis test with Dunn ' s multiple comparison correction ) .

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H) Quantification of KNL‑1 signal intensity at MUC4 foci, normalised to KNL‑1 signal intensity at centromeres (=1, red line). LinesLines = Mean ± SD, 3 experiments, each with ≥ 10 metaphases analysed per condition. Each point = 1 focus or centromere. ns = p>0.05, *** = p<0.01, **** = p<0.001 (Kruskal Wallis test with Dunn's multiple comparison correction).


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . C ) Immunofluorescence images of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against CENP - T . Images in are maximum intensity projections taken across the depth of the EGFP foci .

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. C) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against CENP-T. Images in are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . F ) Immunofluorescence images of CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against KNL - 1 . Images in , F are maximum intensity projections taken across the depth of the EGFP foci .

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. F) Immunofluorescence images of CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against KNL-1. Images in ,F are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . I ) Immunofluorescence images of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted cells stained with antibodies against Ndc80 . Images in I ) are maximum intensity projections taken across the depth of the EGFP foci .

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. I) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against Ndc80. Images in I) are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . A ) Immunofluorescence images of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted metaphase cells stained with antibodies against alpha - tubulin . B ) Immunofluorescence images of mitotic cells with CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeting after nocodazole treatment . C ) Length measurements of individual EGFP foci in µm . Example measurements are indicated on zooms in A - B ) in orange . Data is ≥ 20 cells per condition , from 1 experiment . Lines = Mean ± SEM . * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 001 ( Kruskal - Wallis with multiple comparison correction ) . Images in A , B are maximum intensity projections taken across the depth of the EGFP foci . In A , B ) Orange bars = measured lengths in C .

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. A) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against alpha-tubulin. B) Immunofluorescence images of mitotic cells with CENP‑T∆C‑dCas9‑EGFP targeting after nocodazole treatment. C) Length measurements of individual EGFP foci in µm. Example measurements are indicated on zooms in A-B) in orange. Data is ≥20 cells per condition, from 1 experiment. Lines = Mean ± SEM. * = p<0.05, ** = p<0.01,**** = p<0.001 (Kruskal-Wallis with multiple comparison correction). Images in A,B are maximum intensity projections taken across the depth of the EGFP foci. In A,B) Orange bars = measured lengths in C.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . D ) Immunofluorescence images of metaphase cells with CENP - T ∆ C - dCas9 ‑ EGFP targeting after cold treatment , stained with antibodies against alpha - tubulin . E ) Percentage of metaphase cells showing microtubule ( MT ) recruitment . ≥ 50 metaphases per condition , from 1 experiment . . are maximum intensity projections taken across the depth of the EGFP foci .

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. D) Immunofluorescence images of metaphase cells with CENP-T∆C-dCas9‑EGFP targeting after cold treatment, stained with antibodies against alpha-tubulin. E) Percentage of metaphase cells showing microtubule (MT) recruitment. ≥50 metaphases per condition, from 1 experiment.. are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN in HEK293T cells . F ) Frames from live cell imaging of cells with dCas9 targeted to Chr9 - CEN , or an EGFP negative cell . are maximum intensity projections taken across the depth of the EGFP foci .

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN in HEK293T cells. F) Frames from live cell imaging of cells with dCas9 targeted to Chr9-CEN, or an EGFP negative cell. are maximum intensity projections taken across the depth of the EGFP foci.


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . A ) Immunofluorescence images of paired EGFP foci for each attachment status category . Images were chosen where both the EGFP foci and any of their attachments lay in the same Z - plane for clarity of presentation . B ) Percentage of paired EGFP foci showing each attachment status . are from ≥ 75 EGFP foci pairs from n ≥ 15 metaphases per condition 1 experiment

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. A) Immunofluorescence images of paired EGFP foci for each attachment status category. Images were chosen where both the EGFP foci and any of their attachments lay in the same Z-plane for clarity of presentation. B) Percentage of paired EGFP foci showing each attachment status. are from ≥75 EGFP foci pairs from n≥15 metaphases per condition 1 experiment for


Targeting of dCas9 - EGFP or CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP to Chr9 - CEN and Chr1 ‑ TELO in HEK293T cells . C ) Immunofluorescence images of CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted metaphase cells stained with antibodies against Mad2 . D ) Percentage of individual EGFP foci showing EGFP and Mad2 signal co - localisation , in metaphase cells with CENP ‑ T ∆ C - dCas9 - targeting . 2 experiments for ( D ) . Image in C ) is a maximum intensity projection taken across the depth of the EGFP signal . Scale bars = 1µm on small images and zooms , 5µm on larger images in C ) .

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Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. C) Immunofluorescence images of CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against Mad2. D) Percentage of individual EGFP foci showing EGFP and Mad2 signal co-localisation, in metaphase cells with CENP‑T∆C-dCas9-targeting. 2 experiments for (D). Image in C) is a maximum intensity projection taken across the depth of the EGFP signal. Scale bars = 1µm on small images and zooms, 5µm on larger images in C).


Immunofluorescence images from CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted metaphase cells stained with antibodies against Mad2 and alpha - Tubulin , showing examples of microtubule attached ( G ) EGFP foci . H ) Percentage of individual EGFP foci with EGFP and Mad2 signal co ‑ localisation attached ( H ) EGFP foci . are from ≥ 75 EGFP foci pairs from n ≥ 15 metaphases per condition 1 experiment for H ) are single Z - slices .

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Immunofluorescence images from CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against Mad2 and alpha-Tubulin, showing examples of microtubule attached (G) EGFP foci. H) Percentage of individual EGFP foci with EGFP and Mad2 signal co‑localisation attached (H) EGFP foci. are from ≥75 EGFP foci pairs from n≥15 metaphases per condition 1 experiment for H) are single Z-slices.


C , D ) Example images of chromosome mis - segregation events involving the target locus ( EGFP signal ) in anaphase HEK293T ( C ) or HCT116 ( D ) cells with CENP - T ∆ C - dCas9 ‑ EGFP targeting after Mps1i pulse . E , F ) Mis ‑ segregation rate in fixed dCas9 - targeted HEK293T ( E ) or HCT116 ( F ) cells after Mps1i pulse , involving only EGFP + chromosomes , only EGFP - chromosomes or both . Bars = mean ± SD , 3 experiments for each cell line , each with ≥ 25 anaphase cells per condition . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 ( Oneway ANOVA with Šidák ' s multiple comparison correction , in E / F ) comparing the mis - segregation rate for any events involved EGFP ) .

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C,D) Example images of chromosome mis-segregation events involving the target locus (EGFP signal) in anaphase HEK293T (C) or HCT116 (D) cells with CENP-T∆C-dCas9‑EGFP targeting after Mps1i pulse. E,F) Mis‑segregation rate in fixed dCas9-targeted HEK293T (E) or HCT116 (F) cells after Mps1i pulse, involving only EGFP+ chromosomes, only EGFP- chromosomes or both. Bars = mean ± SD, 3 experiments for each cell line, each with ≥25 anaphase cells per condition. * = p<0.05, **= p<0.01, *** = p<0.001, **** = p<0.0001 (Oneway ANOVA with Šidák's multiple comparison correction, in E/F) comparing the mis-segregation rate for any events involved EGFP).


G - H ) Frames from live cell imaging of HEK293T H2B - RFP cells after treatment with Mps1i ( t = 0 ) , with CENP ‑ T ∆ C ‑ dCas9 ‑ EGFP targeted to Chr9 - CEN or Chr1 - TELO . Frames are either across the duration of the 4 h movie ( G ) or show examples of the different categories of fate ( H ) . I ) Percentage of CENP - T ∆ C - dCas9 ‑ EGFP targeted cells that made an EGFP + error in anaphase , categorised by subsequent fate of EGFP signal in daughter cells 3 h after Mps1i addition . Assessed by live cell imaging . ≥ 22 cells with error per condition , combining data from ≥ 3 independent experiments .

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G-H) Frames from live cell imaging of HEK293T H2B-RFP cells after treatment with Mps1i (t=0), with CENP‑T∆C‑dCas9‑EGFP targeted to Chr9-CEN or Chr1-TELO. Frames are either across the duration of the 4 h movie (G) or show examples of the different categories of fate (H). I) Percentage of CENP-T∆C-dCas9‑EGFP targeted cells that made an EGFP+ error in anaphase, categorised by subsequent fate of EGFP signal in daughter cells 3 h after Mps1i addition. Assessed by live cell imaging. ≥22 cells with error per condition, combining data from ≥3 independent experiments.


( B ) Slice through a tomogram showing EBOV virions inside a late endosomal compartment .

(B) Slice through a tomogram showing EBOV virions inside a late endosomal compartment.


( I - J ) Magnified areas highlighted in ( F ) and ( H ) , respectively , showing the viral membrane and VP40 densities at the luminal side . For comparison , line profiles at 3 nm distance from the inner membrane monolayer , visualized by dotted profiles ( magenta and blue , respectively ) , were determined . ( K ) Line profiles adjacent to the inner viral membrane leaflet of a virion inside an endosome and a purified virion before infection .

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(I-J) Magnified areas highlighted in (F) and (H), respectively, showing the viral membrane and VP40 densities at the luminal side. For comparison, line profiles at 3 nm distance from the inner membrane monolayer, visualized by dotted profiles (magenta and blue, respectively), were determined. (K) Line profiles adjacent to the inner viral membrane leaflet of a virion inside an endosome and a purified virion before infection.


( G - J ) Slices of a tomogram showing a filamentous EBOV VLP composed of VP40 and GP after incubation at low pH ( n = 18 ) . White arrows in ( H ) and ( J ) highlight areas adjacent to the VLP membrane devoid of protein densities in contrast to corresponding slices of VLPs at neutral pH .

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(G-J) Slices of a tomogram showing a filamentous EBOV VLP composed of VP40 and GP after incubation at low pH (n=18). White arrows in (H) and (J) highlight areas adjacent to the VLP membrane devoid of protein densities in contrast to corresponding slices of VLPs at neutral pH.


( K ) Slices of tomograms showing filamentous VLPs composed of VP40 after incubation at neutral ( n = 22 ) and low pH ( n = 8 ) , respectively . ( L ) Line density profiles determined adjacent to the inner membrane monolayer of VLPs incubated at neutral ( blue ) and low pH ( magenta ) . At neutral pH , the VP40 matrix detectable as regular densities in ( D ) have a 5 - 6 nm pitch .

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(K) Slices of tomograms showing filamentous VLPs composed of VP40 after incubation at neutral (n=22) and low pH (n=8), respectively. (L) Line density profiles determined adjacent to the inner membrane monolayer of VLPs incubated at neutral (blue) and low pH (magenta). At neutral pH, the VP40 matrix detectable as regular densities in (D) have a 5-6 nm pitch.


( E ) Free energy profiles of VP40 - lipid interactions at pH 7 . 4 and pH 4 . 5 determined from MD simulations . The plot shows free energy ( in kBT ) at increasing membrane - VP40 distances ( nm ) with indicated three states shown in ( A ) .

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(E) Free energy profiles of VP40-lipid interactions at pH 7.4 and pH 4.5 determined from MD simulations. The plot shows free energy (in kBT) at increasing membrane-VP40 distances (nm) with indicated three states shown in (A).


( F ) EBOV VLP lipid composition showing highly abundant lipids determined by mass spectrometry in mol % . Lipid abbreviations : phosphatidylcholine ( PC ) , phosphatidylserine ( PS ) , phosphatidylethanolamine ( PE ) , phosphatidylinositol ( PI ) , lyso - phosphatidylcholine ( LPC ) , sphingomyelin ( SM ) . Prefix " a " indicates acyl - linked glycerophospholipids , prefix " e " indicates ether - linked ( plasmanyl ) or the presence of one odd and one even chain fatty acyl . Bars represent mean and error bars represent standard error of the mean ( red ) ; n = 3 biological replicates .

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(F) EBOV VLP lipid composition showing highly abundant lipids determined by mass spectrometry in mol%. Lipid abbreviations: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lyso-phosphatidylcholine (LPC), sphingomyelin (SM). Prefix "a" indicates acyl-linked glycerophospholipids, prefix "e" indicates ether-linked (plasmanyl) or the presence of one odd and one even chain fatty acyl. Bars represent mean and error bars represent standard error of the mean (red); n=3 biological replicates.


( B ) Overview confocal fluorescence microscopy image showing pleomorphic pHluorin - labelled VLPs composed of VP40 , pHluorin - VP40 ( ratio 10 : 1 ) and GP . ( C ) Magnified images of representative VLPs acquired during time - lapse microscopy at neutral pH and after acidification to approximately pH 5 . Frames are exemplarily shown at 0 , 1 , 3 , 5 and 10 min after lowering the external pH .

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(B) Overview confocal fluorescence microscopy image showing pleomorphic pHluorin-labelled VLPs composed of VP40, pHluorin-VP40 (ratio 10:1) and GP. (C) Magnified images of representative VLPs acquired during time-lapse microscopy at neutral pH and after acidification to approximately pH 5. Frames are exemplarily shown at 0, 1, 3, 5 and 10 min after lowering the external pH.


( D ) Plot showing the mean relative fluorescence intensities and standard deviation of VLPs imaged at neutral pH , low pH and in the presence of T - X100 at low pH over time . Data was obtained from 3 independent VLP preparations . Number of technical replicates : n = 42 ( pH 7 . 4 ) ; n = 19 ( pH 4 . 5 and pH 5 + T - X100 ) .

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(D) Plot showing the mean relative fluorescence intensities and standard deviation of VLPs imaged at neutral pH, low pH and in the presence of T-X100 at low pH over time. Data was obtained from 3 independent VLP preparations. Number of technical replicates: n=42 (pH 7.4); n=19 (pH 4.5 and pH 5 + T-X100).


( E ) Plot showing the drop of pH inside VLPs over time after lowering the pH of the surrounding buffer to 5 . The dots represent the mean values , and the dashed lines are the theoretical fit to Eq . 3 .

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(E) Plot showing the drop of pH inside VLPs over time after lowering the pH of the surrounding buffer to 5. The dots represent the mean values, and the dashed lines are the theoretical fit to Eq. 3.


( B ) Stalk formation energy as a function of VP40 - membrane interaction energy . The stalk energy for non - interacting VP40 matrix ( u0 = 0 ) is 65 kBT . VP40 matrix layer Young ' s modulus legend - red 5 . 6 MPa , blue 11 . 2 MPa , black 16 . 8 MPa , and orange is infinitely rigid . The bending rigidity ratio between the VP40 matrix layer and lipid monolayer are 1 , 2 , 3 , and infinity , respectively . The line represents an infinitely rigid layer .

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(B) Stalk formation energy as a function of VP40-membrane interaction energy. The stalk energy for non-interacting VP40 matrix (u0 = 0) is 65 kBT. VP40 matrix layer Young's modulus legend - red 5.6 MPa, blue 11.2 MPa, black 16.8 MPa, and orange is infinitely rigid. The bending rigidity ratio between the VP40 matrix layer and lipid monolayer are 1, 2, 3, and infinity, respectively. The line represents an infinitely rigid layer.


( D ) Fusion pore formation energy as a function of VP40 - membrane interaction energy . The discontinuity in the energy is located at the phase line between configurations ( see C ) . The change in pore formation energy , ΔEpore is defined as the difference between fusion - pore formation energy at interaction energy density 0 . 2 kBT / nm2 ( the value found using MD simulations ) to the matrix - free case ( no interaction energy ) . A - D ) Dotted lines serve as a guide to the eye .

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(D) Fusion pore formation energy as a function of VP40-membrane interaction energy. The discontinuity in the energy is located at the phase line between configurations (see C). The change in pore formation energy, ΔEpore is defined as the difference between fusion-pore formation energy at interaction energy density 0.2 kBT/nm2 (the value found using MD simulations) to the matrix-free case (no interaction energy). A-D) Dotted lines serve as a guide to the eye.


( F ) Quantification of FACS data showing EBOV VLP entry as measured by a fluorescence shift of infected cells from emission at 510 nm ( no entry ) to 450 nm ( entry ) . VLPs were treated prior to infection as indicated on the x - axis , with control : uninfected control cells , - : no treatment , T : thermolysin - treatment at neutral pH , Lp : low pH treatment . Target cells were treated with media or ammonium chloride ( NH4Cl ) , n = 3 with 10 ' 000 cells measured per sample .

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(F) Quantification of FACS data showing EBOV VLP entry as measured by a fluorescence shift of infected cells from emission at 510 nm (no entry) to 450 nm (entry). VLPs were treated prior to infection as indicated on the x-axis, with control: uninfected control cells, - : no treatment, T: thermolysin-treatment at neutral pH, Lp: low pH treatment. Target cells were treated with media or ammonium chloride (NH4Cl), n= 3 with 10'000 cells measured per sample.


Immunofluorescence of early pluripotent marker Sall4 in DMSO - and R406 - treated cells on d12 . n = 5 . Scale bar , 100 μm .

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Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in MEFs , intermediate cells on d12 treated with DMSO and R406 , and R1 ( mESCs ) . n = 3 .

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RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3.


Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk . n = 3 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm.


ciPSCs express key pluripotent markers Oct4 , Sox2 and Nanog . Scale bar , 100 μm .

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ciPSCs express key pluripotent markers Oct4, Sox2 and Nanog. Scale bar, 100 μm.


Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12 . n = 6 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca . n = 3 .

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Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3.


Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12 . n = 3 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1 . n = 3 .

Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3.


RT - qPCR analysis of Gldc , Cbs and Tdh expression in R406 - and shSyk - treated cells ( E ) and FK506 - , shPpp3ca - and shNfatc1 - treated cells ( F ) on d8 . n = 3 .

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RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3.


Metabolite abundance of SAM , SAH in samples treated with DMSO and R406 . n = 6 .

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Metabolite abundance of SAM, SAH in samples treated with DMSO and R406. n = 6.


Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12 . n = 3 . Scale bar , 100 μm . Immunofluorescence of Sall4 in NAC - and DPBS - treated cells on d12 . n = 5 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm. Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and HA - treated cells on d12 . n = 3 . RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and NAC - treated cells on d12 . n = 3 .

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

RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.


Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12 . n = 4 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12. n = 4. Scale bar, 100 μm.


Quantification of oxygen consumption rate ( OCR ) of reprogramming intermediates treated with small molecules ( A , C ) and the indicated OCR parameters ( B , D ) . n = 9 .

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Quantification of oxygen consumption rate (OCR) of reprogramming intermediates treated with small molecules (A, C) and the indicated OCR parameters (B, D). n = 9.


Fluorescence images of DCFH - DA staining for ROS levels in cells treated with DMSO , HA , NAC , and R406 on d12 ( E ) and the quantification of the ROS levels ( F ) . n = 4 . Scale bar , 100 μm . MDA quantification in cells treated with DMSO , HA , NAC , and R406 on d12 . n = 9 .

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Fluorescence images of DCFH-DA staining for ROS levels in cells treated with DMSO, HA, NAC, and R406 on d12 (E) and the quantification of the ROS levels (F). n = 4. Scale bar, 100 μm. MDA quantification in cells treated with DMSO, HA, NAC, and R406 on d12. n = 9.


A qRT - PCR analysis of LINC00941 repression during keratinocyte differentiation in primary keratinocyte cultures from three different donors compared to undifferentiated keratinocytes ( D0 ) ( n = 6 biological replicates ) . Data is presented as mean ± SD .

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A qRT-PCR analysis of LINC00941 repression during keratinocyte differentiation in primary keratinocyte cultures from three different donors compared to undifferentiated keratinocytes (D0) (n = 6 biological replicates). Data is presented as mean ± SD.


siPool mediated LINC00941 knockdown ( B ) in calcium - induced keratinocyte differentiation ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool mediated LINC00941 knockdown (B) in calcium-induced keratinocyte differentiation (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool mediated LINC00941 knockdown in calcium - induced keratinocyte differentiation , results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation ( C ) ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool mediated LINC00941 knockdown in calcium-induced keratinocyte differentiation, results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation (C) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures ( D ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures (D) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures results in increased expression of key differentiation genes ( E ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures results in increased expression of key differentiation genes (E) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


F Immunofluorescence analysis shows increased levels of early and late differentiation proteins . Collagen VII ( col VII , green ) stain indicates the basement membrane , nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red ( n = 3 - 4 tissue cultures / knockdown group ; one exemplary picture for each group is depicted ) . Scale bar : 50 µm .

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F Immunofluorescence analysis shows increased levels of early and late differentiation proteins. Collagen VII (col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red (n = 3-4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.


B LINC00941 knockdown efficiency ( siLINC00941 ) in epidermal tissue on day 3 of differentiation as obtained by qRT - PCR measurements ( n = 4 - 5 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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 O O O O O O O O O O O O O O O O O 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 LINC00941 knockdown efficiency (siLINC00941) in epidermal tissue on day 3 of differentiation as obtained by qRT-PCR measurements (n = 4-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


C Heatmap of differentially expressed genes ( padj < 0 . 05 and - 1 > log2FC > 1 ) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes ( n = 4 - 5 epidermal tissue cultures / knockdown group ) .

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C Heatmap of differentially expressed genes (padj < 0.05 and -1 > log2FC > 1) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes (n = 4-5 epidermal tissue cultures/knockdown group).


A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex ( EDC ) ( n = 4 - 5 biological replicates / knockdown group ) .

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A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex (EDC) (n = 4-5 biological replicates/knockdown group).


B LINC00941 knockdown on day 3 in calcium - induced keratinocyte differentiation cultures results in increased expression of SPRR5 ( FC = fold change ) ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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B LINC00941 knockdown on day 3 in calcium-induced keratinocyte differentiation cultures results in increased expression of SPRR5 (FC = fold change) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


C Detected fragments per million ( FPM ) for SPRR5 in control ( siCtrl ) as well as LINC00941 deficient ( siLINC00941 ) epidermal tissue for all biological replicates on day 3 ( D3 ) of differentiation as obtained by DeSeq2 ( FC = fold change ) ( n = 4 ‑ 5 epidermal tissue cultures / knockdown group ) .

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C Detected fragments per million (FPM) for SPRR5 in control (siCtrl) as well as LINC00941 deficient (siLINC00941) epidermal tissue for all biological replicates on day 3 (D3) of differentiation as obtained by DeSeq2 (FC = fold change) (n = 4‑5 epidermal tissue cultures/knockdown group).


SPRR5 induction during the course of keratinocyte ( KC ) differentiation is shown by northern blot ( D ) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes ( n = 8 biological replicates ) . Data is presented as mean ± SD .

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 O O O O O O O O O

SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by northern blot (D) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.


SPRR5 induction during the course of keratinocyte ( KC ) differentiation is shown by qRT - PCR analysis ( E ) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes ( n = 8 biological replicates ) . Data is presented as mean ± SD .

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 O O O O O O O O O O O

SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by qRT-PCR analysis (E) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.


F p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels ( FC = fold change ) ( n = 3 - 4 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels (FC = fold change) (n = 3-4 biological replicates /knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation ( A ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation (A) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT - PCR ( B ) ( FC = fold change ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT-PCR (B) (FC = fold change) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis ( C ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis (C) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


D SPRR5 knockdown efficiency in day 3 ( D3 ) organotypic epidermal tissue ( n = 4 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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D SPRR5 knockdown efficiency in day 3 (D3) organotypic epidermal tissue (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis . Collagen VII ( Col VII , green ) stain indicates the basement membrane , nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red ( n = 4 tissue cultures / knockdown group ; one exemplary picture for each group is depicted ) . Scale bar : 50 µm .

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E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis. Collagen VII (Col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red (n = 4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.


F SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT - PCR ( n = 4 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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F SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT-PCR (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


B Overview of SPRR5 levels in control ( siCtrl ) and SPRR5 deficient ( siSPRR5 ) epidermal tissue on day 3 and day 4 as obtained by qRT - PCR analysis ( n = 3 - 5 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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B Overview of SPRR5 levels in control (siCtrl) and SPRR5 deficient (siSPRR5) epidermal tissue on day 3 and day 4 as obtained by qRT-PCR analysis (n = 3-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


Heatmaps of differentially expressed genes ( padj < 0 . 05 and - 0 . 5 > log2FC > 0 . 5 ) upon SPRR5 depletion on day 3 ( C ) in organotypic epidermal tissue ( n = 3 - 5 epidermal tissue cultures / knockdown group ) .

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Heatmaps of differentially expressed genes (padj < 0.05 and -0.5 > log2FC > 0.5) upon SPRR5 depletion on day 3 (C) in organotypic epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).


Heatmaps of differentially expressed genes ( padj < 0 . 05 and - 0 . 5 > log2FC > 0 . 5 ) upon SPRR5 depletion on day 4 ( D ) in organotypic epidermal tissue ( n = 3 - 5 epidermal tissue cultures / knockdown group ) .

Heatmaps of differentially expressed genes (padj < 0.05 and -0.5 > log2FC > 0.5) upon SPRR5 depletion on day 4 (D) in organotypic epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).


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

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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 . 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 - / - ) .

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

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


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

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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 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 ) .

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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 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 ) .

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

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G. Western blot analysis of subcellular fractions Calnexin, GAPDH, histone H3, hnRNP R and hnRNP A1. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.


H . Western blot analysis of hnRNP R co - immunoprecipitated by an anti - hnRNP A1 antibody from subcellular fractions . Immunoprecipitation with mouse - IgG antibody was used as control .

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H. Western blot analysis of hnRNP R co-immunoprecipitated by an anti-hnRNP A1 antibody from subcellular fractions. Immunoprecipitation with mouse-IgG antibody was used as control.


I . Quantification of 7SK RNA in subcellular fractions by qPCR . Data are mean with SD ; * * P ≤ 0 . 01 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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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 . Quantification of 7SK RNA co - immunoprecipitating with hnRNP A1 from subcellular fractions . Data are mean with SD ; * P ≤ 0 . 05 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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J. Quantification of 7SK RNA co-immunoprecipitating with hnRNP A1 from subcellular fractions. Data are mean with SD; *P ≤ 0.05, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 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 ) .

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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 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 ) .

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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 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 .

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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 . 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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

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


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

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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 . 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 ) .

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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).


Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for the indicated times .

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Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times.


In C endogenous CALCOCO1 is analysed and the bars represent the mean ± sd of band intensities relative to the actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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In C endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for the indicated times . In E , endogenous CALCOCO1 is analysed and the bars represent the mean ± sd of band intensities relative to the actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In E, endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 and immunostained for endogenous GM130 . Scale bars are 5 μm and 2 μm ( zoomed inset ) .

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F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 μm and 2 μm (zoomed inset).


G Same cells as in E were left untreated or treated with Baf A1 for 6 hours , and then immunostained for endogenous p62 and LC3B . Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans .

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G Same cells as in E were left untreated or treated with Baf A1 for 6 hours, and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


H CALCOCO1 , p62 and LC3B puncta in in the indicated conditions , counted using an automated system . The error bars represent mean ± SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * * p < 0 . 001 .

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H CALCOCO1, p62 and LC3B puncta in in the indicated conditions, counted using an automated system. The error bars represent mean±SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.


I Percentage of co - localization of CALCOCO1 puncta with p62 and / or LC3B in cells treated as indicated . The error bars represent mean ± SEM of three independent experiments per condition and over 250 cells per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * * p < 0 . 001 .

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I Percentage of co-localization of CALCOCO1 puncta with p62 and/or LC3B in cells treated as indicated. The error bars represent mean±SEM of three independent experiments per condition and over 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.


J HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1 . Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans .

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J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


A GST pulldown binding assay of in vitro transcribed / translated 35S - Myc - CALCOCO1 with recombinant GST - tagged ATG8 family proteins . GST and GST fusion proteins were visualized by coomassie brilliant blue staining ( bottom panel ) , and the co - precipitated Myc - CALCOCO1 was detected by autoradiography ( upper panel ) . The numbers below the AR represent % binding in the shown AR .

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A GST pulldown binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by coomassie brilliant blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR .


B GST pulldown assay of transiently transfected Myc - CALCOCO1 from HEK293 cell extracts with recombinant GST - tagged ATG8 family proteins . GST and GST fusions were visualized by Ponceau S staining ( bottom panel ) , and co - precipitated Myc - CALCOCO1 detected by immunoblotting with anti - Myc antibody ( upper panel ) .

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B GST pulldown assay of transiently transfected Myc-CALCOCO1 from HEK293 cell extracts with recombinant GST-tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co-precipitated Myc-CALCOCO1 detected by immunoblotting with anti-Myc antibody (upper panel).


GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins . Precipitated GST and GST fusions and co - precipitated Myc - CALCOCO1 constructs were analyzed as in A .

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GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.


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I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST - tagged CALCOCO1 constructs . GST and GST fusions were visualized as in A , and co - precipitated GABARAP with anti - GABARAP antibody ( upper panel ) .

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I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST-tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co-precipitated GABARAP with anti-GABARAP antibody (upper panel).


GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins ( left ) . Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs . The presence of two well separated binding surfaces on ATG8 proteins binding to LIR ( LDS ) and UIR ( UDS ) is indicated ( right ) .

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GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right).


B GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family

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B GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins


C GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 and 35S - Myc - p62 with recombinant GST - GABARAPL2 ( WT and indicated mutants ) .

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C GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 and 35S-Myc-p62 with recombinant GST-GABARAPL2 (WT and indicated mutants).


D GST pulldown assays of in vitro transcribed / translated 35S - Myc - TAX1BP1 ( WT and indicated mutants ) with recombinant GST - GABARAP ( WT and indicated mutants ) .

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D GST pulldown assays of in vitro transcribed/translated 35S-Myc-TAX1BP1 (WT and indicated mutants) with recombinant GST-GABARAP (WT and indicated mutants).


Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 . Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated . The blot panels are from more than one western blot experiment but for clarity , only a single actin / GAPDH loading control is shown . In F , the bars represent the mean ± sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691. Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In F, the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, *p ˂ 0.01; ns is not significant.


G HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B . Scale bars , 5 μm .

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G HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm.


HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 were starved for 4 hours and then immunostained with anti - ATG13 and anti - WIPI2 antibodies . Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans .

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HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were starved for 4 hours and then immunostained with anti-ATG13 and anti-WIPI2 antibodies. Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


In B , the error bars represent mean ± sd of puncta per cell from three independent experiments and100 - 200 cells per each experiment .

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In B, the error bars represent mean±sd of puncta per cell from three independent experiments and100-200 cells per each experiment.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In D the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In D the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In , the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In , the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin ) . In , the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin). In , the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


, I the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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, I the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP - CALCOCO1 in cells extracts from the rescued cells . In the bars represent the mean ± sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP-CALCOCO1 in cells extracts from the rescued cells. In the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


B Co - IP of Myc - VAPA or Myc - VAPB with EGFP - CALCOCO1 from transiently transfected HEK293 cells . The asterisks ( * ) indicate Myc - VAP previously detected on the same

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B Co- IP of Myc-VAPA or Myc-VAPB with EGFP-CALCOCO1 from transiently transfected HEK293 cells. The asterisks (*) indicate Myc-VAP previously detected on the same membrane


C , D GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - VAPA or GST - VAPB constructs . The scheme is a representation of CALCOCO1 domains and motifs .

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C, D GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-VAPA or GST-VAPB constructs.The scheme is a representation of CALCOCO1 domains and motifs.