Accumulation of 2 - 5A and PAR in HME cells pre - incubated with 10 or 100 nM olaparib for 30 min before treatment with H2O2 for 12 min , as visualized by immunostaining with anti - 2 - 5A and anti - PAR . Scale bar , 10μm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O

Accumulation of 2-5A and PAR in HME cells pre-incubated with 10 or 100 nM olaparib for 30 min before treatment with H2O2 for 12 min, as visualized by immunostaining with anti-2-5A and anti-PAR. Scale bar, 10μm.


HME control ( vec ) and OAS single knockout cells ( KO1 - 3 ) , pre - treated with IFNβ for 2 days to induce OAS expression , fixed , and stained with anti - OAS1 , 2 , or 3 . Staining with secondary antibodies is shown as a negative control . Scale bar , 10μm .

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HME control (vec) and OAS single knockout cells (KO1-3), pre-treated with IFNβ for 2 days to induce OAS expression, fixed, and stained with anti-OAS1, 2, or 3. Staining with secondary antibodies is shown as a negative control. Scale bar, 10μm.


HME control cells were treated with IFNβ for 2 days to induce OAS expression , fixed and stained with anti - OAS1 , 2 , or 3 . Scale bar , 10μm .

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HME control cells were treated with IFNβ for 2 days to induce OAS expression, fixed and stained with anti-OAS1, 2, or 3. Scale bar, 10μm.


HME cells expressing Flag - tagged OAS1 , 2 , or 3 were fixed and stained with anti - Flag . Scale bar , 10μm .

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HME cells expressing Flag-tagged OAS1, 2, or 3 were fixed and stained with anti-Flag. Scale bar, 10μm.


Untreated HME ( left ) or PC3 ( right ) cells were stained with anti - OAS1 - 3 and the nuclei were visualized by staining with DAPI . The intensities of OAS1 - 3 fluorescence signals in the nuclei are plotted . Nuclear and cytoplasmic masks were made based on DAPI and phalloidin staining , respectively . Experiments were repeated at least two times ; at least 40 cells per condition were counted . Data are presented as distribution plot with mean value + / - SD ; statistical significance was determined by unpaired Kolmogorov - Smirnov t - test . Scale bar , 10μm .

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Untreated HME (left) or PC3 (right) cells were stained with anti-OAS1-3 and the nuclei were visualized by staining with DAPI. The intensities of OAS1-3 fluorescence signals in the nuclei are plotted. Nuclear and cytoplasmic masks were made based on DAPI and phalloidin staining, respectively. Experiments were repeated at least two times; at least 40 cells per condition were counted. Data are presented as distribution plot with mean value +/- SD; statistical significance was determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.


HME control ( vec ) and KO1 cells were pre - treated with IFNβ for 2 days , followed by a 10 - min treatment with H2O2 . Cytoplasmic , membrane - bound and soluble nuclear fractions of cell lysates were analyzed by the Western method ; staining with anti - AIF and anti - HDAC2 staining was used to characterize membrane and nuclear fractions , respectively . Anti - PAR detects all PAR - modified proteins , which appear as smears .

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HME control (vec) and KO1 cells were pre-treated with IFNβ for 2 days, followed by a 10-min treatment with H2O2. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method; staining with anti-AIF and anti-HDAC2 staining was used to characterize membrane and nuclear fractions, respectively. Anti-PAR detects all PAR-modified proteins, which appear as smears.


HME control ( v ) , KO1 and OAS1hi ( O1hi ) cells were pre - treated with IFNβ for 48 h . Cytoplasmic , membrane - bound and soluble nuclear fractions of cell lysates were analyzed by the Western method . Anti - AIF and anti - HDAC2 were used to characterize membrane and nuclear fractions , respectively ; * indicates lanes where specific signals are detected .

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HME control (v), KO1 and OAS1hi (O1hi) cells were pre-treated with IFNβ for 48 h. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method. Anti-AIF and anti-HDAC2 were used to characterize membrane and nuclear fractions, respectively; * indicates lanes where specific signals are detected.


Representative images and analysis of Western blots for PAR , performed with lysates of HME control ( vec ) OAS single KO cells ( KO1 - 3 ) , treated with H2O2 for 15 or 40 min . β - actin is shown as a loading control . Signal intensity was measured using ImageJ software . Average lane densities , normalized to vector from three independent experiments , are plotted below as mean + SD , * p < 0 . 05 as assessed by Student ' s t - test .

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Representative images and analysis of Western blots for PAR, performed with lysates of HME control (vec) OAS single KO cells (KO1-3), treated with H2O2 for 15 or 40 min. β-actin is shown as a loading control. Signal intensity was measured using ImageJ software. Average lane densities, normalized to vector from three independent experiments, are plotted below as mean + SD, * p < 0.05 as assessed by Student's t-test.


Same as ( A ) , with lysates of HME control ( vec ) cells and cells expressing ectopic OAS1 , OAS2 or OAS3 ( OAS1 - 3hi ) . Experiments were repeated at least three times . Average lane densities normalized to vector are plotted below below as mean + SD , * p < 0 . 05 as assessed by Student ' s t - test .

Same as (A), with lysates of HME control (vec) cells and cells expressing ectopic OAS1, OAS2 or OAS3 (OAS1-3hi). Experiments were repeated at least three times. Average lane densities normalized to vector are plotted below below as mean + SD, * p < 0.05 as assessed by Student's t-test.


HME cells , pre - incubated with IFNβ for 2 days to increase OAS abundance , were treated with H2O2 for 0 - 60 min at 37oC . Western blots of PAR accumulation ( top ) , shift in the position of PARylated PARP1 ( middle ) , OAS2 induction by IFNβ , and β - actin as a loading control are shown . Experiments were repeated twice .

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HME cells, pre-incubated with IFNβ for 2 days to increase OAS abundance, were treated with H2O2 for 0-60 min at 37oC. Western blots of PAR accumulation (top), shift in the position of PARylated PARP1 (middle), OAS2 induction by IFNβ, and β-actin as a loading control are shown. Experiments were repeated twice.


HME control ( vec ) and KO1 cells were pretreated with 0 - 500 IU / ml IFNβ for 2 days , and then treated with 500 μM H2O2 for 10 or 30 min . Western blots of PAR accumulation ( top , high and medium exposure ; middle , low exposure ) , OAS1 - 3 induction by IFNβ , and β - actin as a loading control are shown . The experiment was repeated four times ; ns , non - specific 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 B-CONTROLLED_VAR 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

HME control (vec) and KO1 cells were pretreated with 0-500 IU/ml IFNβ for 2 days, and then treated with 500 μM H2O2 for 10 or 30 min. Western blots of PAR accumulation (top, high and medium exposure; middle, low exposure), OAS1-3 induction by IFNβ, and β-actin as a loading control are shown. The experiment was repeated four times; ns, non-specific band.


HME cells were treated with 750 μM of H2O2 , with and without pre - treatment with olaparib . The accumulation of dead cells was measured with an IncuCyte ZOOM instrument . Means of triplicates ± SD . * * * , p < 0 . 01 in two - way ANOVA . The data represent the results of three experiments .

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HME cells were treated with 750 μM of H2O2, with and without pre-treatment with olaparib. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.01 in two-way ANOVA. The data represent the results of three experiments.


HME control ( vec ) and KO1 - 3 cells were incubated with olaparib for 30 min followed by treatment with 500 μM H2O2 for 15 min . PAR accumulation was assessed by the Western method , with anti - PAR . β - actin is shown as a loading control .

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HME control (vec) and KO1-3 cells were incubated with olaparib for 30 min followed by treatment with 500 μM H2O2 for 15 min. PAR accumulation was assessed by the Western method, with anti-PAR. β-actin is shown as a loading control.


HME vec , KO1 and OAS1hi cells were incubated with olaparib for 30 min , then treated with 500 or 1000 μM of H2O2 and allowed to grow for 72 h . Bars represent means of triplicates ± SD from three experiments . * p < 0 . 05 ; * * p < 0 . 01 ( relative to H2O2 - treated vec cells ) in Student ' s t - test .

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HME vec, KO1 and OAS1hi cells were incubated with olaparib for 30 min, then treated with 500 or 1000 μM of H2O2 and allowed to grow for 72 h. Bars represent means of triplicates ± SD from three experiments. * p<0.05; ** p<0.01 (relative to H2O2-treated vec cells) in Student's t-test.


KO1 cells transfected with vector ( KO1 - vec ) , wild - type OAS1 ( KO1 - OAS1 wt ) , or mutant OAS1 ( D75A / D77A , KO1 - OAS1 mut ) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method . Loading controls : β - actin and overall protein levels were visualized by stain - free technology . The experiment was repeated three times ; a representative image is shown .

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KO1 cells transfected with vector (KO1-vec), wild-type OAS1 (KO1-OAS1 wt), or mutant OAS1 (D75A/D77A, KO1-OAS1 mut) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method. Loading controls: β-actin and overall protein levels were visualized by stain-free technology. The experiment was repeated three times; a representative image is shown.


Quantification of the relative abundance of PAR in ( D ) . Images obtained in three independent experiments were analyzed ; the data are plotted as mean + SD * , p < 0 . 05 by Student ' s t - test .

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Quantification of the relative abundance of PAR in (D). Images obtained in three independent experiments were analyzed; the data are plotted as mean + SD *, p<0.05 by Student's t-test.


KO1 cells transfected with vector , wild - type OAS1 ( OAS1 wt ) , or mutant OAS1 ( D75A / D77A , OAS1 mut ) were treated with 500 μM of H2O2 . The accumulation of dead cells was measured with an IncuCyte ZOOM instrument . Means of triplicates ± SD . * * * * , p < 0 . 001 in two - way ANOVA . The data represent the results of three 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 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

KO1 cells transfected with vector, wild-type OAS1 (OAS1 wt), or mutant OAS1 (D75A/D77A, OAS1 mut) were treated with 500 μM of H2O2. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ****, p<0.001 in two-way ANOVA. The data represent the results of three experiments.


A , B . Representative images and analysis of AIF accumulation in the nuclei of HME control ( vec ) and OAS single - knockout cells ( KO ) , and HME control ( vec ) and OAS double - and triple - knockout cells ( DKOs and TKO ) , 24 h after treatment with 1000 μM H2O2 . Data information : Nuclei were visualized by DAPI staining . Arrowheads : AIF translocated to nuclei . Untreated control cells are shown on the left . Right panels , AIF - positive nuclei were counted from pictures taken from three independent fields in two independent experiments . Means ± SD are plotted . HME : * p < 0 . 005 ; * * p < 0 . 001 compared to H2O2 - treated control ( vec ) cells ;

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

A, B. Representative images and analysis of AIF accumulation in the nuclei of HME control (vec) and OAS single-knockout cells (KO), and HME control (vec) and OAS double- and triple-knockout cells (DKOs and TKO), 24 h after treatment with 1000 μM H2O2. Data information: Nuclei were visualized by DAPI staining. Arrowheads: AIF translocated to nuclei. Untreated control cells are shown on the left. Right panels, AIF-positive nuclei were counted from pictures taken from three independent fields in two independent experiments. Means ±SD are plotted. HME: * p<0.005; ** p<0.001 compared to H2O2-treated control (vec) cells;


C . Representative images and analysis of AIF accumulation in the nuclei of PC3 DKO2 / 3 and PC3 DKO2 / 3 + 1 cells , 24 h after treatment with 100 , 200 or 400 μM H2O2 . Data information : Nuclei were visualized by DAPI staining . Arrowheads : AIF translocated to nuclei . Untreated control cells are shown on the left . Right panels , AIF - positive nuclei were counted from pictures taken from three independent fields in two independent experiments . Means ± SD are plotted . PC3 : * * * p < 0 . 0001 compared to untreated cells of each genotype as assessed by Student ' s t - test . Scale bars , 10μm .

O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images and analysis of AIF accumulation in the nuclei of PC3 DKO2/3 and PC3 DKO2/3+1 cells, 24 h after treatment with 100, 200 or 400 μM H2O2. Data information: Nuclei were visualized by DAPI staining. Arrowheads: AIF translocated to nuclei. Untreated control cells are shown on the left. Right panels, AIF-positive nuclei were counted from pictures taken from three independent fields in two independent experiments. Means ±SD are plotted. PC3: *** p<0.0001 compared to untreated cells of each genotype as assessed by Student's t-test. Scale bars, 10μm.


( A ) Murine Ntras enrichment in endothelial cells versus other heart - derived cell types ( data acquired from GSE95755 ( n = 4 mice ) . Data information : In ( A , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( A ) One - way

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(A) Murine Ntras enrichment in endothelial cells versus other heart-derived cell types (data acquired from GSE95755 (n = 4 mice). Data information: In (A, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (A) One-way ANOVA


( B ) Changes in lncRNA expression from HUVECs exposed to hypoxia ( 0 . 2 % O2 for 12 h or 24 h ) , determined by ribo - minus RNA deep sequencing ( data acquired from GSE107033 ; NTRAS is highlighted ( n = 2 independent biological replicates ) .

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(B) Changes in lncRNA expression from HUVECs exposed to hypoxia (0.2 % O2 for 12 h or 24 h), determined by ribo-minus RNA deep sequencing (data acquired from GSE107033 ; NTRAS is highlighted (n = 2 independent biological replicates).


( C ) Validation of hypoxia - induced NTRAS expression at the indicated time points by RT - qPCR ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - tailed unpaired t -

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(C) Validation of hypoxia-induced NTRAS expression at the indicated time points by RT-qPCR (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test


( D ) Subcellular localization of NTRAS and control transcripts , assayed by nuclear - cytoplasmic fractionation of HUVECs and RT - qPCR ( n = 3 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - tailed unpaired t -

(D) Subcellular localization of NTRAS and control transcripts, assayed by nuclear-cytoplasmic fractionation of HUVECs and RT-qPCR (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test


( E ) FITC - dextran - based in vitro permeability comparing control and NTRAS - silenced HUVECs ( n = 5 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - tailed unpaired t - test ,

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(E) FITC-dextran-based in vitro permeability comparing control and NTRAS-silenced HUVECs (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test,


( F ) Cumulative in vitro sprout lengths of control and NTRAS - silenced HUVECs under basal conditions and VEGFA stimulation ( n = 3 - 10 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(F) Cumulative in vitro sprout lengths of control and NTRAS-silenced HUVECs under basal conditions and VEGFA stimulation (n = 3-10 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( G ) Cell cycle analysis in Ntras - silenced H5V cells ( n = 3 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( G ) two - way ANOVA ,

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(G) Cell cycle analysis in Ntras-silenced H5V cells (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) two-way ANOVA,


( H ) TMR - dextran - based assessment of vascular permeability in vivo , comparing heart homogenates from control and Ntras - silenced mice . Data normalized to organ and body weight ( n = 11 - 12 mice per group ) . Experimental outline on the left . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . H ) two - tailed unpaired t -

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(H) TMR-dextran-based assessment of vascular permeability in vivo, comparing heart homogenates from control and Ntras-silenced mice. Data normalized to organ and body weight (n = 11-12 mice per group). Experimental outline on the left. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. H) two-tailed unpaired t-test


( I ) Survival of control and Ntras - silenced mice under basal conditions ( black , n = 21 - 23 mice per group ) and in hind limb ischemia ( HLI ; red , n = 8 mice per group ) . Number of survivors is indicated ; experimental outline on the left . Data information : ( I ) Mantel - Cox - test .

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(I) Survival of control and Ntras-silenced mice under basal conditions (black, n = 21-23 mice per group) and in hind limb ischemia (HLI; red, n = 8 mice per group). Number of survivors is indicated; experimental outline on the left. Data information: (I) Mantel-Cox-test.


( A ) Separation of mock and proteinase K - treated HeLa nuclear extracts by sucrose density gradient ultracentrifugation followed by NTRAS detection using RT - qPCR . Fractions 1 and 14 represent top and bottom of the gradient , respectively ( n = 1 ) .

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(A) Separation of mock and proteinase K-treated HeLa nuclear extracts by sucrose density gradient ultracentrifugation followed by NTRAS detection using RT-qPCR. Fractions 1 and 14 represent top and bottom of the gradient, respectively (n = 1).


( B ) Enrichment of NTRAS by antisense affinity selection from HeLa cell lysate followed by RT - qPCR , comparing a control and an NTRAS - specific probe ( n = 5 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

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(B) Enrichment of NTRAS by antisense affinity selection from HeLa cell lysate followed by RT-qPCR, comparing a control and an NTRAS-specific probe (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( D ) RT - qPCR - based validation of NTRAS - hnRNPL - interaction by anti - hnRNPL RIPs from HUVEC nuclear fractions ( n = 6 independent biological replicates ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

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(D) RT-qPCR-based validation of NTRAS-hnRNPL-interaction by anti-hnRNPL RIPs from HUVEC nuclear fractions (n = 6 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( E ) RT - qPCR analysis of NTRAS expression in HUVECs upon silencing of hnRNPL ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

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(E) RT-qPCR analysis of NTRAS expression in HUVECs upon silencing of hnRNPL (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( F ) RPLP0 - normalized NTRAS - CA16 motif expression after hnRNPL silencing determined by RT - PCR ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RPLP0-normalized NTRAS-CA16 motif expression after hnRNPL silencing determined by RT-PCR (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( H ) NTRAS and hnRNPL co - regulated alternative splicing ( AS ) events . Data points with an FDR < 0 . 05 are circled ( n = 2 independent 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

(H) NTRAS and hnRNPL co-regulated alternative splicing (AS) events. Data points with an FDR < 0.05 are circled (n = 2 independent biological replicates).


( I ) RT - PCR - based analysis of TJP1 exon 20 inclusion upon silencing of NTRAS in HUVECs ( n = 7 independent biological replicates ) . Representative agarose gels on the right . Data information : , I , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . I , two - tailed unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-PCR-based analysis of TJP1 exon 20 inclusion upon silencing of NTRAS in HUVECs (n = 7 independent biological replicates). Representative agarose gels on the right. Data information: , I, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. I, two-tailed unpaired t-test.


( J ) RT - PCR - based analysis of TJP1 exon 20 inclusion upon silencing of hnRNPL in HUVECs ( n = 4 independent biological replicates ) . Representative agarose gels on the right . Data information : , J ) data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . J ) two - tailed unpaired t - test .

(J) RT-PCR-based analysis of TJP1 exon 20 inclusion upon silencing of hnRNPL in HUVECs (n = 4 independent biological replicates). Representative agarose gels on the right. Data information: , J) data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. J) two-tailed unpaired t-test.


( B ) In vitro splicing efficiency of the TJP1 splice substrate , comparing mock , NTRAS - depleted , and NTRAS - CA16 motif add - back conditions ( n = 7 - 12 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . ( B two - tailed unpaired t - test ,

(B) In vitro splicing efficiency of the TJP1 splice substrate, comparing mock, NTRAS-depleted, and NTRAS-CA16 motif add-back conditions (n = 7-12 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. (B two-tailed unpaired t-test,


( C ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using nuclear lysates from control and NTRAS - silenced HUVECs ( n = 6 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed paired t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-silenced HUVECs (n = 6 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test


( D ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using nuclear lysates from control and NTRAS - overexpressing cells ( n = 5 independent biological replicates ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed paired t - test ,

(D) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test,


( E ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using nuclear lysates from control and NTRAS - CA16 motif overexpressing cells ( n = 5 independent biological replicates ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed paired t - test ,

(E) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-CA16 motif overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test,


( F ) RT - PCR - based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS - CA16 motif ( n = 8 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-PCR-based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS-CA16 motif (n = 8 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. one-way ANOVA.


( G ) Endothelial resistance of NTRAS - silenced HUVECs ( n = 3 - 5 independent biological replicates ) , analyzed by electrical cell - substrate impedance sensing ( ECIS ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t -

(G) Endothelial resistance of NTRAS-silenced HUVECs (n = 3-5 independent biological replicates), analyzed by electrical cell-substrate impedance sensing (ECIS). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test


( H ) Endothelial resistance of hnRNPL - silenced HUVECs ( n = 3 independent biological replicates ) , analyzed by ECIS . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Endothelial resistance of hnRNPL-silenced HUVECs (n = 3 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( I ) Analysis of TJP1 exon 20 inclusion by RT - PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20 - intron 20 boundary ( E20 SSO ) ( n = 9 independent biological replicates ) . Representative agarose gel on the right . Schematic outline at the top right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of TJP1 exon 20 inclusion by RT-PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20 - intron 20 boundary (E20 SSO) (n = 9 independent biological replicates). Representative agarose gel on the right. Schematic outline at the top right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( J ) Endothelial resistance of control SSO - or E20 SSO - transfected HUVECs ( n = 4 independent biological replicates ) , analyzed by ECIS . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Endothelial resistance of control SSO- or E20 SSO-transfected HUVECs (n = 4 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( A ) Validation of Ntras - hnRNPL - interaction by anti - hnRNPL RIPs from H5V lysates , followed by RT - qPCR ( n = 4 independent biological replicates ) . Representative western blot on the right . Data information : In ( A , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( A ) two - tailed paired t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Validation of Ntras-hnRNPL-interaction by anti-hnRNPL RIPs from H5V lysates, followed by RT-qPCR (n = 4 independent biological replicates). Representative western blot on the right. Data information: In (A, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. (A) two-tailed paired t-test


( B ) RT - PCR - based analysis of murine Tjp1 exon 20 inclusion in cardiac tissue from control and Ntras - silenced mice ( n = 8 mice per group ) . Experimental outline on the left . Data information : In data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( B , two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-PCR-based analysis of murine Tjp1 exon 20 inclusion in cardiac tissue from control and Ntras-silenced mice (n = 8 mice per group). Experimental outline on the left. Data information: In data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. (B, two-tailed unpaired t-test,


( D ) Co - precipitation of Ntras in anti - hnRNPL RIPs using whole heart lysates from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice ( n = 3 - 4 mice per group ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-precipitation of Ntras in anti-hnRNPL RIPs using whole heart lysates from NtrasCA/CA and Ntras∆CA/∆CA mice (n = 3-4 mice per group). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( E ) RT - PCR - based analysis of TJP1 exon 20 inclusion in hearts from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice ( n = 9 - 14 mice per group ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-PCR-based analysis of TJP1 exon 20 inclusion in hearts from NtrasCA/CA and Ntras∆CA/∆CA mice (n = 9-14 mice per group). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( F ) Retinal angiogenesis assessed in P7 NtrasCA / CA and Ntras ∆ CA / ∆ CA pups by immunostaining of isolectin B4 . Vascularized areas were normalized to total retinal area ( n = 4 - 8 mice per group ) . Representative micrographs are shown . Scale bars are 200 µm . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Retinal angiogenesis assessed in P7 NtrasCA/CA and Ntras∆CA/∆CA pups by immunostaining of isolectin B4. Vascularized areas were normalized to total retinal area (n = 4-8 mice per group). Representative micrographs are shown. Scale bars are 200 µm. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( G ) FTSC and TMR - dextran in vivo permeability assays , comparing homogenates of hearts from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice . Data normalized to organ and body weight ( n = 11 - 18 mice per group ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FTSC and TMR-dextran in vivo permeability assays, comparing homogenates of hearts from NtrasCA/CA and Ntras∆CA/∆CA mice. Data normalized to organ and body weight (n = 11-18 mice per group). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( H ) Quantification of CD45 + cell ( red ) infiltration into cardiac tissue from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice normalized to DAPI . Isolectin B4 ( green ) was used to label endothelial cells ( n = 4 - 5 mice per group ) . Representative micrographs are shown . Scale bars are 50 µm . Insets and arrows indicate sites of CD45 + cell infiltration . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of CD45+ cell (red) infiltration into cardiac tissue from NtrasCA/CA and Ntras∆CA/∆CA mice normalized to DAPI. Isolectin B4 (green) was used to label endothelial cells (n = 4-5 mice per group). Representative micrographs are shown. Scale bars are 50 µm. Insets and arrows indicate sites of CD45+ cell infiltration. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( I ) Percentage of control and Ntras - silenced mice showing cardiac pathologies ( n = 15 - 17 mice per group ) . Prevalence is indicated above the bars . H & E micrographs of murine heart sections from control and Ntras - silenced mice are shown . Insets and arrows indicate sites of lymphocytic infiltration . Scale bars are 100 µm . Data information : ( I ) Chi - square test .

(I) Percentage of control and Ntras-silenced mice showing cardiac pathologies (n = 15-17 mice per group). Prevalence is indicated above the bars. H&E micrographs of murine heart sections from control and Ntras-silenced mice are shown. Insets and arrows indicate sites of lymphocytic infiltration. Scale bars are 100 µm. Data information: (I) Chi-square test.


( a ) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA - tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA - specific antibody . Data represent mean values ± s . d . ( b ) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins . Data represent mean values ± s . d . ( c ) Relative CK2α protein concentration following injection of proteasome inhibitor ( 1 μM MG132 ) together with unmodified CK2α protein . Data represent mean values ± s . d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA-tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA-specific antibody. Data represent mean values ± s.d. (b) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins. Data represent mean values ± s.d. (c) Relative CK2α protein concentration following injection of proteasome inhibitor (1 μM MG132) together with unmodified CK2α protein. Data represent mean values ± s.d.


( d ) Western blot for total CK2α protein in HeLa cells following treatment with Cdk inhibitors . Numbers indicate relative quantification normalized for loading control . Mean values ( n = 3 ) shown ± s . d .

O O O O O O O O O O O O O O O O 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 for total CK2α protein in HeLa cells following treatment with Cdk inhibitors. Numbers indicate relative quantification normalized for loading control. Mean values (n = 3) shown ± s.d.


( e ) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis . Full gels corresponding to those in panels d and e are in Supplementary Figure 8 .

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

(e) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis. Full gels corresponding to those in panels d and e are in Supplementary Figure 8.


( a ) Pull - down experiments with semisynthetic HA - CK2α protein and recombinant Pin1 . CK2α was immobilized using HA - specific ( anti - HA ) antibodies bound to protein G beads . Pin1 was incubated with immobilized CK2α for 20 min at 4 ° C .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pull-down experiments with semisynthetic HA-CK2α protein and recombinant Pin1. CK2α was immobilized using HA-specific (anti-HA) antibodies bound to protein G beads. Pin1 was incubated with immobilized CK2α for 20 min at 4 °C.


( b ) Coimmunoprecipitation of endogenous Pin1 with CK2α . Pfa - containing and unmodified semisynthetic HA - CK2α proteins were spiked into REF52 cell lysates and immobilized using HA - specific antibodies . Input samples are shown for loading control .

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

(b) Coimmunoprecipitation of endogenous Pin1 with CK2α. Pfa-containing and unmodified semisynthetic HA-CK2α proteins were spiked into REF52 cell lysates and immobilized using HA-specific antibodies. Input samples are shown for loading control.


( c ) Pull - down experiments with Pfa344 HA - CK2α protein and recombinant Pin1 in the presence of Pin1 - specific or nonspecific IgY .

O O O 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) Pull-down experiments with Pfa344 HA-CK2α protein and recombinant Pin1 in the presence of Pin1-specific or nonspecific IgY.


( d ) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1 - specific IgY or nonspecific IgY ( control ) . Data represent mean values ± s . d . Full gels corresponding to those in panels a - c are in Supplementary Figures 10 and 11 .

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 O O O O O O O O O O

(d) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1-specific IgY or nonspecific IgY (control). Data represent mean values ± s.d. Full gels corresponding to those in panels a-c are in Supplementary Figures 10 and 11.


( a ) Phosphorylation of C - terminal tail CK2α peptides ( residues 337 - 352 ) by Cdk1 - cyclin B . In vitro Cdk1 - cyclin B kinase assays were performed using the following peptide substrate : SSMPGGSTPVXSANMMK ( εBiotin ) , where X = serine or S - GlcNAc serine . After reactions were quenched , peptides were separated from the reaction mixture on Tris - tricine gels . Data represent mean values ± s . d .

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

(a) Phosphorylation of C-terminal tail CK2α peptides (residues 337-352) by Cdk1-cyclin B. In vitro Cdk1-cyclin B kinase assays were performed using the following peptide substrate: SSMPGGSTPVXSANMMK(εBiotin), where X = serine or S-GlcNAc serine. After reactions were quenched, peptides were separated from the reaction mixture on Tris-tricine gels. Data represent mean values ± s.d.


( b ) Immunoprecipitation and western blot for O - GlcNAc CK2α in HeLa cells after treatment with the O - GlcNAcase inhibitor TMG for 12 h . Numbers indicate relative quantification normalized for total CK2α concentrations .

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

(b) Immunoprecipitation and western blot for O-GlcNAc CK2α in HeLa cells after treatment with the O-GlcNAcase inhibitor TMG for 12 h. Numbers indicate relative quantification normalized for total CK2α concentrations.


( c ) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h . Numbers indicate relative quantification normalized for loading control , mean values ( n = 4 ) shown ± s . d . Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15 .

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

(c) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h. Numbers indicate relative quantification normalized for loading control, mean values (n = 4) shown ± s.d. Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15.


Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates . All reactions were performed at 30 ° C . Numbers indicate the concentration ( nM ) of product for each kinase reaction . ( a ) AHCYL2 substrate ( 25 ng μl − 1 ) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min . ( b ) NAP1L3 substrate ( 15 ng μl − 1 ) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min . ( c ) NIPBL substrate ( 50 ng μl − 1 ) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min . Full gels corresponding to those in panels a - c are in Supplementary Figures 19 and 25 .

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Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates. All reactions were performed at 30 °C. Numbers indicate the concentration (nM) of product for each kinase reaction. (a) AHCYL2 substrate (25 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (b) NAP1L3 substrate (15 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (c) NIPBL substrate (50 ng μl−1) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min. Full gels corresponding to those in panels a-c are in Supplementary Figures 19 and 25.


F - I ChIP assays with antibodies against H3K9me3 , H4K20me3 , H3K4me3 and H3K9Acetyl were performed as in ( D ) . Only ' C ' position signals were showed and normalized to Input .

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F-I ChIP assays with antibodies against H3K9me3, H4K20me3, H3K4me3 and H3K9Acetyl were performed as in (D). Only 'C' position signals were showed and normalized to Input.


M ChIP assays with antibodies against CDK9 and Pho - Pol II were performed in siNC and siCHAF1A J - Lat 10 . 6 cells . ChIP signals in each group were normalized to Input .

M ChIP assays with antibodies against CDK9 and Pho-Pol II were performed in siNC and siCHAF1A J-Lat 10.6 cells. ChIP signals in each group were normalized to Input.


siRNAs targeting each CAF - 1 subunits and corresponding enriched genes were transfected into TZM - bl cell lines respectively . The fold change of luciferase expression of each groups was normalized to siNC .

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siRNAs targeting each CAF-1 subunits and corresponding enriched genes were transfected into TZM-bl cell lines respectively. The fold change of luciferase expression of each groups was normalized to siNC.


C , D ImmunoFISH images of CAF - 1 bodies and HIV - 1 genomic DNA within J - Lat 10 . 6 . Naïve cells were treated with DMSO . Activated cells were acquired by treating with TNFα .

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C, D ImmunoFISH images of CAF-1 bodies and HIV-1 genomic DNA within J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were acquired by treating with TNFα.


E RFP - tagged CHAF1A and GFP - tagged PML body component SP100 were co - overexpressed in HEK293T cells and imaged by SIM . F GFP - tagged CHAF1A and RFP - tagged BRD4 were co - overexpressed in HEK293T cells and imaged by SIM .

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E RFP-tagged CHAF1A and GFP-tagged PML body component SP100 were co-overexpressed in HEK293T cells and imaged by SIM. F GFP-tagged CHAF1A and RFP-tagged BRD4 were co-overexpressed in HEK293T cells and imaged by SIM.


SUV39H1 , HP1α , DNMT1 , MBD1 , RING1B , EZH2 were co - overexpressed with CHAF1A in HEK293T cells respectively . SIM imaging was performed for each combination .

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SUV39H1, HP1α, DNMT1, MBD1, RING1B, EZH2 were co-overexpressed with CHAF1A in HEK293T cells respectively. SIM imaging was performed for each combination.


E GFP - tagged CHAF1A - N - NLS and CHAF1A - C were imaged with RFP - tagged CHAF1A respectively . N : CHAF1A N - terminal . NLS : nuclear localization signal of CHAF1A , which was presented within C - terminal . C : CHAF1A C - terminal .

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E GFP-tagged CHAF1A-N-NLS and CHAF1A-C were imaged with RFP-tagged CHAF1A respectively. N: CHAF1A N-terminal. NLS: nuclear localization signal of CHAF1A, which was presented within C-terminal. C: CHAF1A C-terminal.


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F Upper panel showed WT-CHAF1A bodies. Lower panel showed dLLPS-CHAF1A which was CHAF1A-Q34A-I116A-K395D-R402D-K409D.


G - I GFP - tagged CHAF1A - IDR and CHAF1A - mIDR were purified in vitro . Ten micromole of each protein was incubated with gradient droplet formation buffer . The droplets of CHAF1A - IDR appeared in NaCl concentration of 125 mM or lower ( G ) . No CHAF1A - mIDR droplet was detected within any NaCl concentration ( I ) . The volumes of CHAF1A - IDR droplets increased with the increase of protein concentration ( H ) . Left subpanels within each panel represented images of protein droplets in different buffers . Right subpanels within each panel represented statistical analysis results of droplets areas and numbers .

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G-I GFP-tagged CHAF1A-IDR and CHAF1A-mIDR were purified in vitro. Ten micromole of each protein was incubated with gradient droplet formation buffer. The droplets of CHAF1A-IDR appeared in NaCl concentration of 125 mM or lower (G). No CHAF1A-mIDR droplet was detected within any NaCl concentration (I). The volumes of CHAF1A-IDR droplets increased with the increase of protein concentration (H). Left subpanels within each panel represented images of protein droplets in different buffers. Right subpanels within each panel represented statistical analysis results of droplets areas and numbers.


B First panel : the co - localization of PCNA and CHAF1A . Second panel : the distribution of PCNA in the absence of endogenous CHAF1A . Third panel : the distribution of PCNA in the presence of CHAF1A - dPIP1 mutant and the absence of endogenous CHAF1A . Fourth panel : the co - localization of PCNA and over - expressed DNMT1 in the absence of endogenous CHAF1A .

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B First panel: the co-localization of PCNA and CHAF1A. Second panel: the distribution of PCNA in the absence of endogenous CHAF1A. Third panel: the distribution of PCNA in the presence of CHAF1A-dPIP1 mutant and the absence of endogenous CHAF1A. Fourth panel: the co-localization of PCNA and over-expressed DNMT1 in the absence of endogenous CHAF1A.


C Upper two panels : the distribution of HP1α in the presence or absence of CHAF1A . Lower two panels : the distribution of HP1α in the presence of CHAF1A - dHP1BD or CHAF1A - DS2 - dHP1BD . Both images of lower two panels were in the absence of endogenous CHAF1A .

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C Upper two panels: the distribution of HP1α in the presence or absence of CHAF1A. Lower two panels: the distribution of HP1α in the presence of CHAF1A-dHP1BD or CHAF1A-DS2-dHP1BD. Both images of lower two panels were in the absence of endogenous CHAF1A.


The distribution of SUV39H2 ( E ) and MBD1 ( F ) in the presence or absence of CHAF1A .

The distribution of SUV39H2 (E) and MBD1 (F) in the presence or absence of CHAF1A.


G The distribution of SUMO paralogs in the absence of CHAF1A .

G The distribution of SUMO paralogs in the absence of CHAF1A.


A Flow cytometry figures of CAF - 1 body rescue experiment . First figure : Wildtype J - Lat 10 . 6 . Second figure : CHAF1A knock - out J - Lat 10 . 6 . Third figure : CHAF1A - KO J - Lat 10 . 6 was re - introduced with wildtype CHAF1A . Fourth figure : CHAF1A - KO J - Lat 10 , 6 was re - introduced with dLLPS - CHAF1A which harbored five mutations . The percentages of GFP - positive cells were labeled in the top right corner of each figure .

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A Flow cytometry figures of CAF-1 body rescue experiment. First figure: Wildtype J-Lat 10.6. Second figure: CHAF1A knock-out J-Lat 10.6. Third figure: CHAF1A-KO J-Lat 10.6 was re-introduced with wildtype CHAF1A. Fourth figure: CHAF1A-KO J-Lat 10,6 was re-introduced with dLLPS-CHAF1A which harbored five mutations. The percentages of GFP-positive cells were labeled in the top right corner of each figure.


D The amounts of intracellular HIV - 1 RNAs of latently infected primary CD4 + T cells which were isolated from PBMCs of HIV - 1 - infected individuals . αCD3 / αCD28 / IL - 2 treatment was performed as positive control . shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A . shluc lentiviruses treatment was performed as negative control . SAHA was added as LRA supplement .

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D The amounts of intracellular HIV-1 RNAs of latently infected primary CD4+ T cells which were isolated from PBMCs of HIV-1-infected individuals. αCD3/αCD28/IL-2 treatment was performed as positive control. shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A. shluc lentiviruses treatment was performed as negative control. SAHA was added as LRA supplement.


B . Before differentiation , hESCs show uniformly high expression of OCT4 . Treatment with BMP4 produces a mixture of OCT4 + / CDX2 - self - renewing hESCs and OCT4 - / CDX2 + mesodermal cells .

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B. Before differentiation, hESCs show uniformly high expression of OCT4. Treatment with BMP4 produces a mixture of OCT4+/CDX2- self-renewing hESCs and OCT4-/CDX2+ mesodermal cells.


C . Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs . Cells were assigned to one of two distinct populations based on a 2 - component mixed Gaussian

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C. Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs. Cells were assigned to one of two distinct populations based on a 2-component mixed Gaussian distribution


E . Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration . Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity .

E. Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration. Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity.


F . Distribution of OCT4 levels in individual hESCs . A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell .

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F. Distribution of OCT4 levels in individual hESCs. A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell.


G . Single - cell traces of OCT4 signaling . The length of each cell ' s trace indicates its cell cycle duration .

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G. Single-cell traces of OCT4 signaling. The length of each cell's trace indicates its cell cycle duration.


H . Distribution of cell cycle durations for 120 hESCs .

H. Distribution of cell cycle durations for 120 hESCs.


A . Single - cell traces of hESCs before and after treatment with 100 ng / mL BMP4 . Cells were imaged for 42 h prior to BMP4 treatment . Mean nuclear OCT4 levels were quantified every 5 minutes and individual cells were tracked from the cell division event that created the cell until its own division .

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A. Single-cell traces of hESCs before and after treatment with 100 ng/mL BMP4. Cells were imaged for 42 h prior to BMP4 treatment. Mean nuclear OCT4 levels were quantified every 5 minutes and individual cells were tracked from the cell division event that created the cell until its own division.


B . 24 h after BMP4 treatment , cells were fixed , stained for expression of CDX2 , and returned to the microscope for registration with the final time - lapse image . Mean nuclear OCT4 - mCherry and CDX2 were quantified for each cell , and the resulting distribution was fit to a 2 - component mixed Gaussian distribution representing self - renewing ( OCT4 + / CDX2 - ) and differentiated ( OCT4 - / CDX2 + ) cells hESCs that could be assigned to either distribution with > 99 % confidence ( red and green dots , Pdiff < 0 . 01 or Pdiff > 0 . 99 ) were considered for pro - fate analysis . Cells that did not reach this threshold ( gray dots ) were not used to determine pro - fate . Because cells in the final frame were assigned to only self - renewing or differentiated categories , pro - mixed

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B. 24 h after BMP4 treatment, cells were fixed, stained for expression of CDX2, and returned to the microscope for registration with the final time-lapse image. Mean nuclear OCT4-mCherry and CDX2 were quantified for each cell, and the resulting distribution was fit to a 2-component mixed Gaussian distribution representing self-renewing (OCT4+/CDX2-) and differentiated (OCT4-/CDX2+) cells hESCs that could be assigned to either distribution with >99% confidence (red and green dots, Pdiff < 0.01 or Pdiff > 0.99) were considered for pro-fate analysis. Cells that did not reach this threshold (gray dots) were not used to determine pro-fate. Because cells in the final frame were assigned to only self-renewing or differentiated categories, pro-mixed cells


C . Distributions of OCT4 levels in pro - self - renewing , pro - mixed , and pro - differentiated cell populations .

C. Distributions of OCT4 levels in pro-self-renewing, pro-mixed, and pro-differentiated cell populations.


D . Distributions of OCT4 pulse frequencies in pro - pluripotent , pro - mixed , and pro - differentiated cell populations . To gain an unbiased look at preexisting determinants of cell fate in panels only cells who completed their entire cell cycle duration before BMP4 addition ( t = 0 ) were included in the analysis .

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D. Distributions of OCT4 pulse frequencies in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis.


E . Distributions of cell cycle durations in pro - pluripotent , pro - mixed , and pro - differentiated cell populations . To gain an unbiased look at preexisting determinants of cell fate in panels , only cells who completed their entire cell cycle duration before BMP4 addition ( t = 0 ) were included in the analysis .

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E. Distributions of cell cycle durations in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels , only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis.


F . Decision surfaces for logistic regression classifier . Cells with self - renewing ( red ) or differentiated ( green ) pro - fates are plotted above the predicted class surfaces . ( left ) Slice of decision surface for pulse frequency vs . OCT4 levels at a cell cycle duration of 14 h . ( right ) Slice of decision surface for cell cycle duration vs . OCT4 level at a pulse frequency of 0 . 25 h - 1 . * P < 0 . 05 , * * P < 0 . 0005 ; ns , not significant .

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F. Decision surfaces for logistic regression classifier. Cells with self-renewing (red) or differentiated (green) pro-fates are plotted above the predicted class surfaces. (left) Slice of decision surface for pulse frequency vs. OCT4 levels at a cell cycle duration of 14 h. (right) Slice of decision surface for cell cycle duration vs. OCT4 level at a pulse frequency of 0.25 h-1. * P < 0.05, ** P < 0.0005; ns, not significant.


A . Lineage of OCT4 expression dynamics . Mean nuclear OCT4 levels were quantified in individual hESCs continuously for 72 h under undifferentiated conditions . Vertical bars represent individual cells that completed an entire cell cycle lifetime . Thin horizontal bars denote cell division events . Color scale indicates low ( black ) , intermediate ( red ) , and high ( white ) OCT4 expression levels .

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A. Lineage of OCT4 expression dynamics. Mean nuclear OCT4 levels were quantified in individual hESCs continuously for 72 h under undifferentiated conditions. Vertical bars represent individual cells that completed an entire cell cycle lifetime. Thin horizontal bars denote cell division events. Color scale indicates low (black), intermediate (red), and high (white) OCT4 expression levels.


B . Differences in OCT4 levels between sister cells , cousin cells , second cousin cells , and randomly paired cells .

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B. Differences in OCT4 levels between sister cells, cousin cells, second cousin cells, and randomly paired cells.


C . Filmstrip showing distribution of OCT4 to daughter cells during cell division .

C. Filmstrip showing distribution of OCT4 to daughter cells during cell division.


D . Distribution of OCT4 ratios between sister cells before and after BMP4 treatment . Ratios for both sister cells ( r and 1 / r ) are plotted to emphasize central tendency toward r = 1 : 1 . Differently colored curves represent the distribution of ratios at different time points after division . For time points after 5 minutes , the ratio was determined by first calculating a mean OCT4 level for each sister cell among all previous time points and then calculating the resulting ratio between sisters .

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D. Distribution of OCT4 ratios between sister cells before and after BMP4 treatment. Ratios for both sister cells (r and 1/r) are plotted to emphasize central tendency toward r = 1:1. Differently colored curves represent the distribution of ratios at different time points after division. For time points after 5 minutes, the ratio was determined by first calculating a mean OCT4 level for each sister cell among all previous time points and then calculating the resulting ratio between sisters.


E . Proliferating H9 - OCT4 - mCherry hESCs were imaged for 24 h , fixed , and stained for DNA content with DAPI . The probability density of DAPI ratio ( blue ) and OCT4 - mCherry ratio ( red ) between sister cells that divided ≤ 1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function . A two - sample Kolmogorov - Smirnov test was used to determine significance . * P < 0 . 05 , * * * P < 0 . 0005 , two - sample Kolmogorov - Smirnov test ; ns , not significant .

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E. Proliferating H9-OCT4-mCherry hESCs were imaged for 24 h, fixed, and stained for DNA content with DAPI. The probability density of DAPI ratio (blue) and OCT4-mCherry ratio (red) between sister cells that divided ≤1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function. A two-sample Kolmogorov-Smirnov test was used to determine significance. * P < 0.05, *** P < 0.0005, two-sample Kolmogorov-Smirnov test; ns, not significant.


B . OCT4 mixing as a function of cell history . 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division .

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B. OCT4 mixing as a function of cell history. 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division.


C . Variance in final OCT4 expression explained over time during maternal , inherited , and autonomous periods of cell history . At each time point , the coefficient of determination ( R2 ) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated .

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C. Variance in final OCT4 expression explained over time during maternal, inherited, and autonomous periods of cell history. At each time point, the coefficient of determination (R2) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated.


D . Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells . The same R2 over time mother - daughter traces were randomly sampled from a set of mother - daughter traces that contained sister - cell pairs ( blue line ) or only one randomly selected sister cell from each pair ( red line ) . E . Difference in variance explained between red and blue traces in panel D . The black line represents the actual difference ; the red line shows a smoothing ( moving average = 1 . 25 h ) of the black trace to highlight the overall trend .

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D. Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells. The same R2 over time mother-daughter traces were randomly sampled from a set of mother-daughter traces that contained sister-cell pairs (blue line) or only one randomly selected sister cell from each pair (red line). E. Difference in variance explained between red and blue traces in panel D. The black line represents the actual difference; the red line shows a smoothing (moving average = 1.25 h) of the black trace to highlight the overall trend.


Western blot analysis of soluble and insoluble fractions ( Red - and yellow rectangles , highlights CTF - 35 and CTF - 25 respectively in the insoluble fraction ) .

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Western blot analysis of soluble and insoluble fractions (Red- and yellow rectangles, highlights CTF-35 and CTF-25 respectively in the insoluble fraction).


Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP - 43 ( pTDP - 43 ) . Scale bar : 20µm . Presented immunofluorescent images of control ( C2 ) and mutant TDP - 43 ( P2 ) . I represents its corresponding profile intensity plot of the selected ( square ) region . Scale bar : 10µm , J quantification pooled control ( C0 = 10 , C1 = 10 and C2 = 10 ) n = 30 and pooled mutant TDP - 43 ( P1 = 10 , P2 = 10 and P3 = 10 ) n = 30 cells from each differentiation . Unpaired Mann - Whitney - test .

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Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP-43 (pTDP-43). Scale bar: 20µm. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). I represents its corresponding profile intensity plot of the selected (square) region. Scale bar: 10µm, J quantification pooled control (C0=10, C1=10 and C2=10) n=30 and pooled mutant TDP-43 (P1=10, P2=10 and P3=10) n=30 cells from each differentiation. Unpaired Mann-Whitney-test.


Western blots showing nucleo - cytoplasmic fractionation of WT - mCherry and MUT - mCherry iPSC - derived motor neurons with F ( left panel ) quantification of total mCherry and total untagged TDP - 43 on α - tubulin and ( right panel ) relative cytoplasmic mCherry and untagged TDP - 43 on total mCherry and total untagged TDP - 43

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Western blots showing nucleo-cytoplasmic fractionation of WT-mCherry and MUT-mCherry iPSC-derived motor neurons with F (left panel) quantification of total mCherry and total untagged TDP-43 on α-tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP-43 on total mCherry and total untagged TDP-43 respectively


Representative kymographs from control and mutant TDP - 43 iPSC - derived motor neurons . The x - axis represent distance in µm while on the y - axis time in seconds is revealed . Stationary mitochondria are visible as straight vertical lines , while moving mitochondria are depicted as skewed lines . B Quantification of percentage of moving mitochondria .

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Representative kymographs from control and mutant TDP-43 iPSC-derived motor neurons. The x-axis represent distance in µm while on the y-axis time in seconds is revealed. Stationary mitochondria are visible as straight vertical lines, while moving mitochondria are depicted as skewed lines. B Quantification of percentage of moving mitochondria.


Immunofluorescent analysis showing fluorescence intensity of pTDP - 43 in motor neurons . Scale bar : 10µm and its perspective intensity plot in F . G Quantification of E , each dot represents one analysed cell , unpaired Mann - Whitney - test .

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Immunofluorescent analysis showing fluorescence intensity of pTDP-43 in motor neurons. Scale bar: 10µm and its perspective intensity plot in F. G Quantification of E, each dot represents one analysed cell, unpaired Mann-Whitney-test.


axonal transport analysis . Quantification of percentage of moving mitochondria ( J ) , number of moving mitochondria ( K ) and number of stationary mitochondria ( L ) normalized to neurite length 100µm .

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axonal transport analysis. Quantification of percentage of moving mitochondria (J), number of moving mitochondria (K) and number of stationary mitochondria (L) normalized to neurite length 100µm.


Total cell lysates were fractionated into soluble and insoluble fractions and analysed by Western blot , representative Western blots of soluble and insoluble fraction without C and with D HDAC6 inhibitor treatment .

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Total cell lysates were fractionated into soluble and insoluble fractions and analysed by Western blot, representative Western blots of soluble and insoluble fraction without C and with D HDAC6 inhibitor treatment.


Immunofluorescent analysis of TDP - 43 in control , mutant TDP - 43 and mutant TDP - 43 + HDAC6 inhibitor after 12 h treatment . Presented immunofluorescent images of control ( C2 ) and mutant TDP - 43 ( P2 ) . Scale bar : 25µm . B Profile intensity plot of control , mutant TDP - 43 and mutant TDP - 43 + HDAC6 inhibitor . C Quantification of nucleo - cytoplasmic ratio ( N / C ) fluorescent intensity of TDP - 43 in motor neurons , each dot represents one analyzed cell , unpaired Mann - Whitney - test .

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Immunofluorescent analysis of TDP-43 in control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor after 12 h treatment. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). Scale bar: 25µm. B Profile intensity plot of control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor. C Quantification of nucleo-cytoplasmic ratio (N/C) fluorescent intensity of TDP-43 in motor neurons, each dot represents one analyzed cell, unpaired Mann-Whitney-test.


Quantification of relative nuclear mCherry TDP - 43 on total fraction , ratio paired - t - test .

Quantification of relative nuclear mCherry TDP-43 on total fraction, ratio paired-t-test.


Quantification of percentage of moving mitochondria ,

Quantification of percentage of moving mitochondria,


( C ) Representative IHC images of CD3 + T cells and Iba1 + macrophages / MG in the isotype and αCD4 + αCD8 group . Scale bar ; 100 μm . ( D ) Quantification of Iba1 + and CD3 + cells in IHC sections of BrM from the isotype ( n = 9 ) and αCD4 + αCD8 group ( n = 7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative IHC images of CD3+ T cells and Iba1+ macrophages/MG in the isotype and αCD4+αCD8 group. Scale bar; 100 μm. (D) Quantification of Iba1+ and CD3+ cells in IHC sections of BrM from the isotype (n=9) and αCD4+αCD8 group (n=7).


( F ) Kaplan - Meier curves depict BrM free survival of mice in the isotype and αCD4 + αCD8 group ( Isotype n = 19 , αCD4 + αCD8 n = 19 ) .

(F) Kaplan-Meier curves depict BrM free survival of mice in the isotype and αCD4+αCD8 group (Isotype n=19, αCD4+αCD8 n=19).


( H ) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL - CD4 and TIL - CD8 compared to blood lymphocytes . Values display the average per group based on vst values ( BL - CD4 or BL - CD8 with n = 3 for each group ) vs . BrM - associated TIL - CD4 or TIL - CD8 ( n = 3 for each group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL-CD4 and TIL-CD8 compared to blood lymphocytes. Values display the average per group based on vst values (BL-CD4 or BL-CD8 with n=3 for each group) vs. BrM-associated TIL-CD4 or TIL-CD8 (n=3 for each group).


( A ) Surface expression of PD - 1 in vivo in 99LN - BrM quantified by flow cytometry ( n = 3 ) . ( B ) PD - L1 in 99LN BrM quantified by flow cytometry ( n = 3 ) . ( C ) Flow cytometric analysis of PD - L1 in tumor - free brain and 99LN - BrM ( n = 3 ) . ( D ) Flow cytometric analysis of PD - L1 expression on 99LN - BrM associated my myeloid cell types . ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Surface expression of PD-1 in vivo in 99LN-BrM quantified by flow cytometry (n=3). (B) PD-L1 in 99LN BrM quantified by flow cytometry (n=3). (C) Flow cytometric analysis of PD-L1 in tumor-free brain and 99LN-BrM (n=3). (D) Flow cytometric analysis of PD-L1 expression on 99LN-BrM associated my myeloid cell types. (n=6).


( H ) Representative HALO spatial plot showing the spatial distribution of cell types with indicated marker combination in 99LN - BrM and representative IF images of multiplexed histology . Scale bar ; 100 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

(H) Representative HALO spatial plot showing the spatial distribution of cell types with indicated marker combination in 99LN-BrM and representative IF images of multiplexed histology. Scale bar; 100 μm.


( C ) Quantification of Iba1 + macrophages in IHC sections of 99LN - BrM at trial endpoint ( Ctrl n = 27 , WBRT n = 18 ) . ( D ) Representative IF images of 99LN - BrM stained for the macrophage marker Iba1 ( red ) and the MG marker Tmem119 ( white ) . Higher magnification images present areas in the peri - tumor region and in the tumor core . DAPI was used as nuclear counterstain . Scale bar ; 100 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

(C) Quantification of Iba1+ macrophages in IHC sections of 99LN-BrM at trial endpoint (Ctrl n=27, WBRT n=18). (D) Representative IF images of 99LN-BrM stained for the macrophage marker Iba1 (red) and the MG marker Tmem119 (white). Higher magnification images present areas in the peri-tumor region and in the tumor core. DAPI was used as nuclear counterstain. Scale bar; 100 µm.


( G ) Quantification of CD11c + MHCII + immune cells by flow cytometry in 99LN - BrM with and without WBRT . ( H , I ) Relative proportion of cDC1 and cDC2 in 99LN - BrM with and without WBRT . ( J ) cDC1 : cDC2 ratio in 99LN - BrM based on flow cytometry data ( H + I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of CD11c+MHCII+ immune cells by flow cytometry in 99LN-BrM with and without WBRT. (H, I) Relative proportion of cDC1 and cDC2 in 99LN-BrM with and without WBRT. (J) cDC1:cDC2 ratio in 99LN-BrM based on flow cytometry data (H+I).


( K ) Representative IHC images of CD3 + , FoxP3 + and CD8 + T cells in 99LN - BrM sections with and without WBRT . Scale bar ; 100 µm . ( L ) Quantification of CD3 + cells in IHC sections of 99LN - BrM . ( Ctrl n = 26 , WBRT n = 18 ) . ( M ) FoxP3 : CD3 ratio in IHC sections of 99LN - BrM . ( Ctrl n = 25 , WBRT n = 18 ) . ( N ) CD8 : CD3 ratio in IHC sections of 99LN - BrM . ( Ctrl n = 25 , WBRT n = 18 ) .

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

(K) Representative IHC images of CD3+, FoxP3+ and CD8+ T cells in 99LN-BrM sections with and without WBRT. Scale bar; 100 µm. (L) Quantification of CD3+ cells in IHC sections of 99LN-BrM. (Ctrl n=26, WBRT n=18). (M) FoxP3:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18). (N) CD8:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18).


( B ) Percent T cells of estimated cells in BrM and CLN of mice from control or WBRT group .

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

(B) Percent T cells of estimated cells in BrM and CLN of mice from control or WBRT group.


( B ) Representative MRI images of 99LN - BrM from mice at different time points after treatment start .

(B) Representative MRI images of 99LN-BrM from mice at different time points after treatment start.


( G ) Kaplan - Meier curves show the percentage of symptom - free mice .

(G) Kaplan-Meier curves show the percentage of symptom-free mice.


( K ) Relative Gzmb and IFNγ protein level on CD4 + and CD8 + T cells cultivated with different cell types , with or without aPD - 1 and unstimulated or stimulated with tumor conditioned media ( Cond . ) ( n = 4 for conditions including BMDM , n = 5 for all other conditions ) .

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

(K) Relative Gzmb and IFNγ protein level on CD4+ and CD8+ T cells cultivated with different cell types, with or without aPD-1 and unstimulated or stimulated with tumor conditioned media (Cond.) (n=4 for conditions including BMDM, n=5 for all other conditions).


( a ) Knockdown of RARα in NIH3T3 mouse fibroblasts was conducted using two different shRNAs , sh1 and sh2 , compared to control ( Ctr ) . Left , representative immunoblot . Actin is shown as loading control and full - length blots are shown in Supplementary Figure 21 . Right , amounts of RARα in control and knockdown cells determined by densitometric quantification of immunoblots represented by the one shown on the left . Values are normalized for actin and expressed as multiples of control ( None ) values ; n = 3 .

(a) Knockdown of RARα in NIH3T3 mouse fibroblasts was conducted using two different shRNAs, sh1 and sh2, compared to control (Ctr). Left, representative immunoblot. Actin is shown as loading control and full-length blots are shown in Supplementary Figure 21. Right, amounts of RARα in control and knockdown cells determined by densitometric quantification of immunoblots represented by the one shown on the left. Values are normalized for actin and expressed as multiples of control (None) values; n = 3.


( b ) Rates of degradation of long - lived proteins in control and RARα ( - ) cells maintained in the presence or absence of serum for 12 h . Values are expressed as percentage of proteolysis ; n = 3 .

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

(b) Rates of degradation of long-lived proteins in control and RARα(-) cells maintained in the presence or absence of serum for 12 h. Values are expressed as percentage of proteolysis; n = 3.


( c , d ) Percentage of degradation due to lysosomes ( c ) and macroautophagy ( d ) in cells assayed as in b but treated with inhibitors of lysosomal proteolysis ( c ) or with 3 - methyladenine ( 3MA ) to block macroautophagy ( d ) . Values are expressed as percentage of total protein degradation sensitive to the lysosomal inhibitors ; n = 3 . In all panels , all values are mean ± s . e . m . , and differences with control are significant for * P 0 . 05 .

O O O O O O O O O O O O O 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 O B-CONTROLLED_VAR 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 O O O O 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,d) Percentage of degradation due to lysosomes (c) and macroautophagy (d) in cells assayed as in b but treated with inhibitors of lysosomal proteolysis (c) or with 3-methyladenine (3MA) to block macroautophagy (d). Values are expressed as percentage of total protein degradation sensitive to the lysosomal inhibitors; n = 3. In all panels, all values are mean ± s.e.m., and differences with control are significant for *P 0.05.


( a ) Immunoblot for LC3 - II in control mouse fibroblasts ( Ctr ) or those knocked down for RARα ( RARα ( − ) ) maintained in the presence or absence of serum for the indicated times . Where indicated , protease inhibitors ( PI ) against lysosomal proteolysis were added . Actin is shown as a loading control . ' I ' and ' II ' designate different forms of LC3 , as described in the text . ( b ) Amounts of LC3 - II determined by densitometric quantification of immunoblots . Values are expressed as fold change in value relative to serum - supplemented control cells ; n = 4 . ( c ) Ratio of the amounts of LC3 - II in cells treated with protease inhibitors compared to untreated cells ( None ) . Values are expressed as fold change in value relative to untreated cells ; n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot for LC3-II in control mouse fibroblasts (Ctr) or those knocked down for RARα (RARα(−)) maintained in the presence or absence of serum for the indicated times. Where indicated, protease inhibitors (PI) against lysosomal proteolysis were added. Actin is shown as a loading control. 'I' and 'II' designate different forms of LC3, as described in the text. (b) Amounts of LC3-II determined by densitometric quantification of immunoblots. Values are expressed as fold change in value relative to serum-supplemented control cells; n = 4. (c) Ratio of the amounts of LC3-II in cells treated with protease inhibitors compared to untreated cells (None). Values are expressed as fold change in value relative to untreated cells; n = 4.


( d , e ) Autophagic flux in the same cells as in c expressing mCherry - GFP - LC3 - II and maintained in the presence or absence of serum . Shown in d are representative merged channel images ; scale bars , 2 μm . Arrows indicate autolysosomes ( red ) . Shown in e are the quantification of the number of autophagosomes ( mCherry and GFP positive ) per cell ( left ) and percentage of autolysosomes ( mCherry positive and GFP negative ; right ) in > 50 cells in at least four different fields . AV , autophagic vacuoles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,e) Autophagic flux in the same cells as in c expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged channel images; scale bars, 2 μm. Arrows indicate autolysosomes (red). Shown in e are the quantification of the number of autophagosomes (mCherry and GFP positive) per cell (left) and percentage of autolysosomes (mCherry positive and GFP negative; right) in >50 cells in at least four different fields. AV, autophagic vacuoles.


( f ) Control and RARα ( - ) cells were transfected with the KFERQ - mCherry1 photoactivatable reporter , and after photoactivation they were maintained in medium with or without serum . Left , representative images . Graph shows quantification of the number of puncta per cell in > 50 cells using the CMA reporter in at least four different fields . Nuclei are labeled with DAPI . Scale bars , 10 μm . In b , c , e and f , all values are mean ± s . e . m . Differences with control ( marked with asterisk ) or with serum - supplemented cells ( marked with § ) are significant for P 0 . 05 . Full - field fluorescence images and full - length blots are shown in Supplementary Figures 2 and 21 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Control and RARα(-) cells were transfected with the KFERQ-mCherry1 photoactivatable reporter, and after photoactivation they were maintained in medium with or without serum. Left, representative images. Graph shows quantification of the number of puncta per cell in >50 cells using the CMA reporter in at least four different fields. Nuclei are labeled with DAPI. Scale bars, 10 μm. In b, c, e and f, all values are mean ± s.e.m. Differences with control (marked with asterisk) or with serum-supplemented cells (marked with §) are significant for P 0.05. Full-field fluorescence images and full-length blots are shown in Supplementary Figures 2 and 21, respectively.


( a ) Rates of degradation of long - lived proteins in mouse fibroblasts untreated ( None ) or treated with 40 μM ATRA and maintained in the presence or absence of serum . Values are expressed as a percentage of proteolysis ; n = 3 .

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

(a) Rates of degradation of long-lived proteins in mouse fibroblasts untreated (None) or treated with 40 μM ATRA and maintained in the presence or absence of serum. Values are expressed as a percentage of proteolysis; n = 3.


( b ) Percentage of lysosomal degradation calculated after treatment with inhibitors of lysosomal proteolysis for 12 h ; n = 3 .

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

(b) Percentage of lysosomal degradation calculated after treatment with inhibitors of lysosomal proteolysis for 12 h; n = 3.


( c ) Immunoblot for LC3 - II of the same cells as in a and b maintained in the presence or absence of serum and protease inhibitors ( PI ) . Top , representative immunoblot . Actin is shown as loading control . Bottom , ratio of amount of LC3 - II in cells treated with PI to that in untreated cells . Values are expressed as fold change in value relative to untreated cells ; n = 4 . ' I ' and ' II ' designate different forms of LC3 , as described in the text .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot for LC3-II of the same cells as in a and b maintained in the presence or absence of serum and protease inhibitors (PI). Top, representative immunoblot. Actin is shown as loading control. Bottom, ratio of amount of LC3-II in cells treated with PI to that in untreated cells. Values are expressed as fold change in value relative to untreated cells; n = 4. 'I' and 'II' designate different forms of LC3, as described in the text.


( d , e ) Autophagic flux in untreated and ATRA - treated cells expressing mCherry - GFP - LC3 - II and maintained in the presence or absence of serum . Shown in d are representative merged - channel images . Arrows indicate autolysosomes ( red ) . Scale bar , 2 μm . Shown in e are the number of autophagosomes ( left ) and percentage of autolysosomes ( right ) after quantification of > 50 cells . AV , autophagic vacuoles .

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 O O O O O O

(d,e) Autophagic flux in untreated and ATRA-treated cells expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged-channel images. Arrows indicate autolysosomes (red). Scale bar, 2 μm. Shown in e are the number of autophagosomes (left) and percentage of autolysosomes (right) after quantification of >50 cells. AV, autophagic vacuoles.


( f ) Mouse fibroblasts expressing the KFERQ - mCherry1 photoactivatable reporter with or without ATRA and after photoactivation maintained in the presence or absence of serum . Left , representative images . Nuclei are labeled with DAPI . Scale bars , 10 μm . Graph shows quantification of the number of puncta per cell in > 50 cells . All values in a - c , e and f are mean ± s . e . m . , and differences with untreated ( marked with asterisk ) or with serum - supplemented cells ( marked with § ) are significant for P 0 . 01 . Full - field fluorescence images and full - length blots are shown in Supplementary Figures 3 and 21 , respectively .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter with or without ATRA and after photoactivation maintained in the presence or absence of serum. Left, representative images. Nuclei are labeled with DAPI. Scale bars, 10 μm. Graph shows quantification of the number of puncta per cell in >50 cells. All values in a-c, e and f are mean ± s.e.m., and differences with untreated (marked with asterisk) or with serum-supplemented cells (marked with §) are significant for P 0.01. Full-field fluorescence images and full-length blots are shown in Supplementary Figures 3 and 21, respectively.