( D ) Same as in ( B ) except MEFs were co - transduced with lentiviruses expressing possible pairwise combinations of shRNAs for nine TRs . Left : Heat map depicting effects of paired KD combinations in Reprogramming Efficiency ( RE % ) . Shown are average REs values from at least two independent experiments . REs of double KDs were evaluated against RE of corresponding single KDs . Right : Alkaline Phosphatase ( AP ) stained cultures of terminal reprogrammed MEFs upon representative single double KDs of our shRNA - screens .

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(D) Same as in (B) except MEFs were co-transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs. Left: Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency (RE%). Shown are the average REs values from at least two independent experiments. The REs of the double KDs were evaluated against the RE of the corresponding single KDs. Right: Alkaline Phosphatase (AP) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA-based screens.


( A ) in situ hybridization depicting expression patterns of nine TRs in days 3 6 of MEFs reprogramming . boundaries of early - iPSCs formations are indicated by a blue dashed . Scale bars : 100 μm .

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(A) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming. The boundaries of the early-iPSCs formations are indicated by a blue dashed line. Scale bars: 100 μm.


( C ) Bar graph showing of isolated from early - iPSCs colonies or outside of colonies ( of population ) expressing each of 9 TRs . Data are shown as mean ± of at least two independent experiments . 0 . 05 < ( * ) p < 0 . 1 , * p < 0 . 05 , * * p < 0 . 01 by unpaired Student ' s t - or Welch ' s t - , when appropriate .

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(C) Bar graph showing the percentage of cells isolated from early-iPSCs colonies or outside of the colonies (rest of population) expressing each of the 9 TRs. Data are shown as mean ± SEM of at least two independent experiments. 0.05 < (*)p < 0.1, *p < 0.05, **p < 0.01 by unpaired Student's t-test or Welch's t-test, when appropriate.


( D ) Scatter plot depicting a visual unbiased representation of percentile of expressing each of nine TRs . Each dot depicts of expressing indicated TRs in experiments . Data are shown as mean ± of at least two independent experiments . Anova p < 0 . 0001 .

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(D) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs. Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments. Data are shown as mean ± SEM of at least two independent experiments. Anova test p<0.0001.


( E ) Schematic representation of statistical estimating probability of co - expression of nine TRs per within early - iPSCs colonies . Each row consists of 9 tiles representing a single each column shows expression status of each of nine TRs ( shown with different color ) in 282 ( right side legend ) . co - expression of gradually increasing numbers of TRs is grouped from bottom to top of figure . left part has been generated by estimating percentages of expression of any single TR per as derived from single gene expression in single , while right part depicts co - expression of combinations of nine TRs on double triple single - experiments .

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(E) Schematic representation of the statistical analysis estimating the probability of co-expression of the nine TRs per cell within the early-iPSCs colonies. Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs (shown with different color) in 282 cells (right side legend). The co-expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure. The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells, while the right part depicts the co-expression of combinations of the nine TRs based on double and triple single-cell experiments.


( A - I ) Shown are ChIP - seq big wig files in IGV browser depicting binding of Oct4 ( Red ) , Sox2 ( Blue ) , Klf4 ( Green ) c - Myc ( Black ) to putative regulatory regions in nine TRs genes in MEFs undergoing reprogramming ( 18 hrs , D3 D5 ) in control MEFs , mESCs miPSCs . peaks have been normalized against input . relative sizes of represented genomic loci corresponding TSSs are also indicated . side color bar depicts expression of each TR at indicated time points .

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(A-I) Shown are ChIP-seq big wig files in the IGV browser depicting the binding of Oct4 (Red), Sox2 (Blue), Klf4 (Green) and c-Myc (Black) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming (18 hrs, D3 and D5) and in control MEFs, mESCs and miPSCs. All peaks have been normalized against input DNA. The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated. The side color bar depicts the expression levels of each TR at the indicated time points.


( C ) summarizing effects of each of nine TRs single knockdowns on expression of other TRs on day 6 ; color denotes no change in expression .

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(C) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6; The white color denotes no change in expression levels.


( D ) Scatter plots depicting extent of co - expression of either broadly - or early - iPSCs - specifically expressed TRs in obtained from early - iPSCs colonies or from of population ( left right panel respectively ) . Each dot depicts of co - expressing a unique combination of two or three TRs . Data are shown as mean ± of at least two independent experiments .

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(D) Scatter plots depicting the extent of co-expression of either the broadly- or early-iPSCs-specifically expressed TRs in individual cells obtained from the early-iPSCs colonies or from the rest of the population (left and right panel respectively). Each dot depicts the percentage of cells co-expressing a unique combination of two or three TRs. Data are shown as mean ± SEM of at least two independent experiments.


( E ) summarizing most striking effects of double nine TRs KDs on expression of other TRs on expression of Epcam Lin28a pluripotency markers ; color indicates no change in expression . genes examined have been ranked in according to their order of upregulation during reprogramming ; Lin28a is latest upregulated gene .

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(E) Heatmap summarizing the most striking effects of double nine TRs KDs on the expression of the other TRs and on the expression of the Epcam and Lin28a pluripotency markers; the white color indicates no change in expression levels. The genes examined have been ranked in the heatmap according to their order of upregulation during reprogramming; Lin28a is the latest upregulated gene.


( B ) Shown is a bar graph depicting effect of overexpression of indicated TRs in reprogramming efficiency . Data are shown as mean ± of at least two independent experiments . y axis labeled as % reprogramming efficiency refers to AP + colonies scored as iPSCs .

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(B) Shown is a bar graph depicting the effect of overexpression of the indicated TRs in reprogramming efficiency. Data are shown as mean ± SEM of at least two independent experiments. The y axis labeled as % reprogramming efficiency refers to the AP+ colonies scored as iPSCs.


( C ) Shown is a bar graph depicting effects of rescue experiments in reprogramming efficiency . MEFs undergoing reprogramming were knocked down using shRNAs for endogenous Nanog or Irf6 expression were co - infected with a vector expressing an shRNA - resistant human homologue of Nanog or Irf6 ( hNanog OE , hIrf6 OE ) . Data are shown as mean ± of two independent experiments .

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(C) Shown is a bar graph depicting the effects of rescue experiments in reprogramming efficiency. MEFs undergoing reprogramming were knocked down using shRNAs for the endogenous Nanog or Irf6 expression and were co-infected with a vector expressing an shRNA-resistant human homologue of Nanog or Irf6 (hNanog OE, hIrf6 OE). Data are shown as mean ± SEM of two independent experiments.


B , Immunolocalization of TSN2 - TAPa GFP - TAPa fusion in root of 5 - d - old seedlings . seedlings were grown under no stress ( NS ) conditions ( 23ºC ) or incubated for 40 min at 39ºC ( HS ) then immunostained with α - Myc . Scale bars = 10 μm .

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B, Immunolocalization of TSN2-TAPa and GFP-TAPa fusion proteins in root cells of 5-d-old seedlings. The seedlings were grown under no stress (NS) conditions (23ºC) or incubated for 40 min at 39ºC (HS) and then immunostained with α-Myc. Scale bars = 10 μm.


B , Co - localization of RFP - TSN2 ( red ) with GFP - ADH2 ( negative control ) GFP - UBP1 ( positive control ) in N . benthamiana protoplasts incubated under 23 ° C ( NS ) or at 39ºC for 40 min ( HS ) . Scale bars = 5 μm .

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B, Co-localization of RFP-TSN2 (red) with GFP-ADH2 (negative control) and GFP-UBP1 (positive control) in N. benthamiana protoplasts incubated under 23°C (NS) or at 39ºC for 40 min (HS). Scale bars = 5 μm.


C , Pearson Spearman coefficients ( rp rs , respectively ) of co - localization ( PSC ) of RFP - TSN2 with GFP - tagged TSN - interacting listed in A with both negative positive control ( denoted by red arrowheads ) under HS . Data represent means ± SD of at least 5 replicate measurements from three independent experiments .

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C, Pearson and Spearman coefficients (rp and rs, respectively) of co-localization (PSC) of RFP-TSN2 with individual GFP-tagged TSN-interacting proteins listed in A and with both negative and positive control proteins (denoted by red arrowheads) under HS. Data represent means ± SD of at least 5 replicate measurements from three independent experiments.


D , BiFC between cYFP - TSN2 nYFP - TSN - interacting in N . benthamiana leaf or protoplasts after HS ( 39ºC for 40 min ) . BiFC of cYFP - TSN2 nYFP - TSN - interacting ( TIPs ) with empty vectors ( EV ) encoding nYFP cYFP , respectively was used as a negative control . Only one representative example of BiFC between cYFP nYFP - TIP is shown . Scale bars = 5 μm .

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D, BiFC between cYFP-TSN2 and nYFP-TSN-interacting proteins in N. benthamiana leaf cells or protoplasts after HS (39ºC for 40 min). BiFC analysis of cYFP-TSN2 and nYFP-TSN-interacting proteins (TIPs) with empty vectors (EV) encoding nYFP and cYFP, respectively was used as a negative control. Only one representative example of BiFC between cYFP and nYFP-TIP is shown. Scale bars = 5 μm.


D , BiFC between cYFP - TSN2 ( full - ) , cYFP - SN or cYFP - Tudor nYFP - TSN - interacting in N . benthamiana protoplasts after HS ( 39ºC for 40 min ) . Scale bars = 10 μm . Boxplots show of reconstituted YFP signal . AU , arbitrary units . Upper lower box boundaries represent first third quantiles , respectively , horizontal lines mark median , whiskers mark highest lowest values . Three independent experiments , each containing seven measurements , were performed . * * * P < 0 . 001 vs Tudor ( one - way ANOVA ) .

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D, BiFC between cYFP-TSN2 (full-length), cYFP-SN or cYFP-Tudor and nYFP-TSN-interacting proteins in N. benthamiana protoplasts after HS (39ºC for 40 min). Scale bars = 10 μm. Boxplots show quantification of the reconstituted YFP signal. AU, arbitrary units. Upper and lower box boundaries represent the first and third quantiles, respectively, horizontal lines mark the median, and whiskers mark the highest and lowest values. Three independent experiments, each containing seven individual measurements, were performed. ***P < 0.001 vs Tudor (one-way ANOVA).


B , Co - immunoprecipitation ( Co - Ip ) of two TSN isoforms RH12 in extracts prepared from N . benthamiana leaves agro - infiltrated with GFP - TSN1 or GFP - TSN2 Myc - RH12 . Free GFP was used as a negative control . Input Co - Ip fractions were analyzed by immunoblotting using α - Myc α - GFP .

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B, Co-immunoprecipitation (Co-Ip) of the two TSN isoforms and RH12 in protein extracts prepared from N. benthamiana leaves agro-infiltrated with GFP-TSN1 or GFP-TSN2 and Myc-RH12. Free GFP was used as a negative control. Input and Co-Ip fractions were analyzed by immunoblotting using α-Myc and α-GFP.


C , Localization of RH12 in root of 5 - d - old Arabidopsis seedlings expressing GFP - RH12 under control of native promoter . seedlings were grown under 23 ° C ( NS ) or incubated at 39 ° C for 60 min ( HS ) . Scale bars = 10 μm .

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C, Localization of RH12 in root cells of 5-d-old Arabidopsis seedlings expressing GFP-RH12 under control of the native promoter. The seedlings were grown under 23°C (NS) or incubated at 39°C for 60 min (HS). Scale bars = 10 μm.


B , Co - immunoprecipitation ( Co - Ip ) of TSN RBP47 in extracts prepared from 10 - d - old Arabidopsis seedlings expressing Pro35S : GFP - RBP47 grown under no stress ( NS ) , HS ( 39ºC for 60 min ) or salt ( NaCl ) stress ( 150 mM NaCl for 60 min ) conditions . GFP - expressing was used as a negative control . Endogenous TSN ( 107 kD ) was detected in total fractions ( Input ) fractions co - immunoprecipitated ( Co - Ip ) with RBP47 but not with free GFP in three conditions . Input Co - Ip fractions were analyzed by immunoblotting using α - TSN α - GFP .

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B, Co-immunoprecipitation (Co-Ip) of TSN and RBP47 in protein extracts prepared from 10-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47 and grown under no stress (NS), HS (39ºC for 60 min) or salt (NaCl) stress (150 mM NaCl for 60 min) conditions. The GFP-expressing line was used as a negative control. Endogenous TSN (107 kD) was detected in total fractions (Input) and fractions co-immunoprecipitated (Co-Ip) with RBP47 but not with free GFP in all three conditions. Input and Co-Ip fractions were analyzed by immunoblotting using α-TSN and α-GFP.


C , Localization of GFP - tagged in root of 5 - d - old Arabidopsis seedlings expressing Pro35S : GFP - RBP47 , Pro35S : GFP - UBP1 , Pro35S : GFP - TCTP ProUBQ : GFP - SnRK1α2 . seedlings were grown under 23 ° C ( NS ) , incubated at 39 ° C for 60 min ( HS ) or treated with 200 mM NaCl at 23 ° C for 60 min ( NaCl ) . Scale bars = 10

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C, Localization of GFP-tagged proteins in root cells of 5-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47, Pro35S:GFP-UBP1, Pro35S:GFP-TCTP and ProUBQ:GFP-SnRK1α2. The seedlings were grown under 23°C (NS), incubated at 39°C for 60 min (HS) or treated with 200 mM NaCl at 23°C for 60 min (NaCl). Scale bars = 10 μm


D , Localization of GFP - SnRK1α1 GFP - SnRK1α2 in root of 5 - d - old Arabidopsis WT tsn1 tsn2 seedlings grown under 23 ° C ( NS ) or incubated at 39 ° C for 60 min ( HS ) . Insets show enlarged areas inside dashed rectangles . Scale bars = 10 μm .

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D, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT and tsn1 tsn2 seedlings grown under 23°C (NS) or incubated at 39°C for 60 min (HS). Insets show enlarged areas inside dashed rectangles. Scale bars = 10 μm.


J , Colocalization of GFP - SnRK1α1 , GFP - SnRK1α1CD , or GFP - SnRK1α1RD with RFP - RBP47 in N . benthamiana protoplasts subjected to HS ( 40 min at 39 ° C ) . For colocalization under NS conditions see Fig . EV5B . For CHX treatment , protoplasts were incubated with 200 ng μL - 1 CHX for 30 min at 23ºC before HS . GFP RFP fusion were expressed under control of UBQ 35S promoter , respectively . Scale bars = 5 μm .

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J, Colocalization of GFP-SnRK1α1, GFP-SnRK1α1CD, or GFP-SnRK1α1RD with RFP-RBP47 in N. benthamiana protoplasts subjected to HS (40 min at 39°C). For colocalization analysis under NS conditions see Fig. EV5B. For CHX treatment, protoplasts were incubated with 200 ng μL-1 CHX for 30 min at 23ºC before HS. GFP and RFP fusion proteins were expressed under the control of the UBQ and 35S promoter, respectively. Scale bars = 5 μm.


C , Localization of GFP - SnRK1α1 GFP - SnRK1α2 in root of 5 - d - old Arabidopsis WT seedlings incubated at 39 ° C imaged at indicated time points . Scale bars = 10 μm . D , of GFP - SnRK1α1 GFP - SnRK1α2 foci in experiment shown in C . Data represent means ± SD of at least 16 replicate measurements . experiment was repeated three times with similar results . * * * P < 0 . 05 ( two - t - ) .

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C, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT seedlings incubated at 39°C and imaged at the indicated time points. Scale bars = 10 μm. D, Quantification of GFP-SnRK1α1 and GFP-SnRK1α2 foci in the experiment shown in C. Data represent means ± SD of at least 16 replicate measurements. The experiment was repeated three times with similar results. ***P < 0.05 (two-tailed t-test).


E , F , Immunoblot with indicated antibodies of extracts prepared from root tips of 10 - d - old Arabidopsis tsn1 tsn2 ( E ) or tsn1 tsn2 expressing ProTSN2 : GFP - TSN2 ( F ) heat - stressed seedlings ( 39ºC ) 0 , 20 , 40 60 min after onset of HS . charts show SnRK1 activity , expressed as of phosphorylated to total SnRK1 . data represent mean ratios of integrated band intensities ( for both isoforms ) normalized to 0 min ± SD from at least four different experiments . P values denote statistically significant differences for comparisons to 0 min ( two - t - ) .

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E, F, Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10-d-old Arabidopsis tsn1 tsn2 (E) or tsn1 tsn2 expressing ProTSN2:GFP-TSN2 (F) heat-stressed seedlings (39ºC) 0, 20, 40 and 60 min after the onset of HS. The charts show SnRK1 activity, expressed as the ratio of phosphorylated to total SnRK1 protein. The data represent mean ratios of integrated band intensities (for both isoforms) normalized to 0 min ± SD from at least four different experiments. P values denote statistically significant differences for comparisons to 0 min (two-tailed t-test).


G , Expression of DIN2 DIN6 in Arabidopsis WT , tsn1 tsn2 , tsn1 tsn2 ; TSN2 snrk1α1 - / - snrk1α2 - / + 10 - d - old heat - stressed seedlings relative to unstressed controls . For CHX treatment , WT seedlings were pre - treated with 200 ng μL - 1 CHX for 30 min before HS . Upper lower box boundaries represent first third quantiles , respectively . Horizontal lines mark median of five replicate measurements , whiskers mark highest lowest values . Means with different letters are significantly different at P < 0 . 05 ( one - way ANOVA ) .

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

G, Expression levels of DIN2 and DIN6 in Arabidopsis WT, tsn1 tsn2, tsn1 tsn2;TSN2 and snrk1α1-/- snrk1α2-/+ 10-d-old heat-stressed seedlings relative to unstressed controls. For CHX treatment, the WT seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. Upper and lower box boundaries represent the first and third quantiles, respectively. Horizontal lines mark the median of five replicate measurements, and whiskers mark the highest and lowest values. Means with different letters are significantly different at P < 0.05 (one-way ANOVA).


( D ) HeLa were transfected with 10 nM control ( n = 84 ) or Nup50 siRNA ( n = 171 ) , 20 nM control ( n = 101 ) or Nup50 siRNA ( n = 157 ) 40 nM control ( n = 86 ) or Nup50 siRNA ( n = 204 ) . 72 h post transfection , were fixed with 4 % PFA stained with antibodies against Nup50 mAB414 , was labeled with DAPI . Scale bars : 50 µm . Quantitation of mAB414 rim at three different Nup50 RNAi control oligo concentrations . means are indicated as diamonds , error bars show standard deviations . P - values have been calculated from a student t - comparing mean between experimental conditions .

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(D) HeLa cells were transfected with 10 nM control (n=84) or Nup50 siRNA (n=171), 20 nM control (n=101) or Nup50 siRNA (n=157) and 40 nM control (n=86) or Nup50 siRNA (n=204). 72 h post transfection, cells were fixed with 4% PFA and stained with antibodies against Nup50 and mAB414, chromatin was labeled with DAPI. Scale bars: 50 µm. Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations. The means are indicated as diamonds, error bars show the standard deviations. P-values have been calculated from a student t-test comparing the mean between the experimental conditions.


( A ) Mouse 3T3 - NIH were transfected with 20 nM control , Nup50A , Nup50B or a combination of Nup50A Nup50B siRNA . After 72h were fixed stained with mAB414 ( green ) antibodies against mouse Nup50 ( red ) . is stained with DAPI ( blue ) . merge of three channels is shown on left column . Scale bar : 50 µm . An insert shows a zoom on a representative nucleus for each picture . Scale bar : 10 µm .

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(A) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B siRNA. After 72h cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative nucleus for each picture. Scale bar: 10 µm.


( B ) Quantitation of mAB414 rim from n = 4 independent experiments done as in ( A ) , each represented by a different color . mean of each experiment condition is represented by a diamond , mean of four experiments by a horizontal black . P - values have been calculated from a paired t - comparing mean between experimental conditions . Error bars show standard deviations of means .

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(B) Quantitation of the mAB414 rim intensity from n=4 independent experiments done as in (A), each represented by a different color. The mean of each individual experiment and condition is represented by a diamond, the mean of all four experiments by a horizontal black line. P-values have been calculated from a paired ratio t-test comparing the mean between the experimental conditions. Error bars show the standard deviations of the means.


( A , B ) Demembranated sperm was preincubated in Xenopus egg extract , depleted for MEL28 / ELYS or Nup50 ( B ) or control treated ( Mock , A ) . After 10 min , membranes were added to reaction . Reactions were stopped at indicated time points by fixation analyzed by confocal microscopy after immunostaining with α - MEL28 / ELYS ( A ) , α - Nup50 ( B ) , mAb414 ( A , B ) . was stained with DAPI ( blue in overlay ) . Scale bar : 10 μm .

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(A, B) Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B) or control treated (Mock, A). After 10 min, membranes were added to the reaction. Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α-MEL28/ELYS (A), α-Nup50 (B), and mAb414 (A, B). Chromatin was stained with DAPI (blue in the overlay). Scale bar: 10 μm.


3 µM recombinant EGFP , EGFP - tagged MEL28 / ELYS ( aa 2290 - 2408 ) or EGFP - tagged Nup50 were incubated with empty or - coated magnetic beads , which were chromatinized with Xenopus egg extracts ( right panel ) or unchromatinized - beads ( left panel ) . After 3h beads were re - isolated , washed , co - stained with DAPI analyzed by confocal microscopy . Scale bar : 10 µm .

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3 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or EGFP-tagged Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA-beads (left panel). After 3h the beads were re-isolated, washed, co-stained with DAPI and analyzed by confocal microscopy. Scale bar: 10 µm.


( C ) HeLa were transfected with EGFP - Nup50 comprising different point mutations in minimal NPC binding region analyzed as in ( A ) . Scale Bar : 10 µm .

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(C) HeLa cells were transfected with EGFP-Nup50 comprising different point mutations in the minimal NPC binding region and analyzed as in (A). Scale Bar: 10 µm.


( D ) GST - fusion constructs of Xenopus Nup50 minimal NPC binding fragment ( aa 144 - 189 ) comprising no or single point mutations were incubated with Xenopus egg extracts . Starting material as well as bound were analyzed by Western blotting with indicated antibodies . quantitation shows average MEL28 / ELYS Nup153 bead bound signal from three independent experiments , normalized to signal of their respective wild - type GST fusion Data points from experiments are indicated .

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(D) GST-fusion constructs of the Xenopus Nup50 minimal NPC binding fragment (aa 144-189) comprising no or single point mutations were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies. The quantitation shows the average MEL28/ELYS and Nup153 bead bound signal from three independent experiments, normalized to the signal of their respective wild-type GST fusion Data points from individual experiments are indicated.


( A ) Confocal microscopy of nuclei assembled for 120 min in mock depleted , Nup50 depleted ( ∆ Nup50 ) , Nup50 depleted Xenopus egg extracts supplemented with recombinant wild - type Nup50 or different mutants . Nuclei were fixed in 4 % PFA 0 . 5 % glutaraldehyde , stained for NPCs ( mAB414 ) ( DAPI ) . Scale bar : 10 µm . ( B ) Average of mAB414 positive nuclei for 100 randomly chosen substrates in each of at least three independent experiments shown in ( A ) . Data points from experiments are indicated .

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(A) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants. Nuclei were fixed in 4% PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. (B) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated.


( C ) Confocal microscopy of nuclei assembled with N - terminal Nup50 truncations an minimal N - terminal fragment . Samples were analyzed as in ( A ) . ( D ) Average of mAB414 positive nuclei for 100 randomly chosen substrates in each of at least three independent experiments shown in ( C ) . Data points from experiments are indicated .

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(C) Confocal microscopy images of nuclei assembled with N-terminal Nup50 truncations and an minimal N-terminal fragment. Samples were analyzed as in (A). (D) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (C). Data points from the individual experiments are indicated.


( B ) Confocal microscopy of fixed nuclei assembled for 120 min in mock depleted ( mock ) Nup50 depleted ( ∆ Nup50 ) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologues . Nuclei were stained for Nup50 ( Nup50 antibody for Xenopus , His6 for mouse ) NPCs ( mAB414 , red ) on ( DAPI ) . Scale bar : 10 µm . ( C ) average of mAB414 positive nuclei for 100 randomly chosen substrates in each of three independent experiments ( performed as in B ) is shown . Data points from experiments are indicated .

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(B) Confocal microscopy images of fixed nuclei assembled for 120 min in mock depleted (mock) and Nup50 depleted (∆Nup50) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologues. Nuclei were stained for Nup50 (Nup50 antibody for Xenopus protein, His6 for mouse protein) and NPCs (mAB414, red) on the chromatin (DAPI). Scale bar: 10 µm. (C) The average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments (performed as in B) is shown. Data points from individual experiments are indicated.


( E ) GST - fusion constructs of Xenopus Nup53 RRM domain ( aa 162 - 267 , control ) full - Xenopus Nup50 two mouse Nup50 orthologues were incubated with Xenopus egg extracts . Starting material as well as bound were analyzed by Western blotting with indicated antibodies right panel shows average MEL28 / ELYS , Nup153 , importin β ran bead bound signal from two independent experiments normalized to their respective Xenopus signal . Data points from experiments are indicated .

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(E) GST-fusion constructs of the Xenopus Nup53 RRM domain (aa 162-267, control) full-length Xenopus Nup50 and the two mouse Nup50 orthologues were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies The right panel shows the average MEL28/ELYS, Nup153, importin β and ran bead bound signal from two independent experiments normalized to their respective Xenopus signal. Data points from individual experiments are indicated.


HEK293T were transfected with empty FLAG - tag vector as a control or FLAG - Nup50 N - terminal fragment ( aa 1 - 120 of human ) . 24 h post transfection were lysed , FLAG - tagged immuno - isolated analyzed by mass spectrometry with Volcano blot showing identified interactors of Nup50 ( yellow , data from three independent experiments ) Data points from experiments are indicated . KPNA2 is gene name of importin α .

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HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1-120 of the human sequence). 24 h post transfection cells were lysed, FLAG-tagged proteins immuno-isolated and analyzed by mass spectrometry with the Volcano blot showing the identified interactors of Nup50 (yellow, data from three independent experiments) Data points from individual experiments are indicated. KPNA2 is the gene name of importin α.


HEK293T were transfected with empty FLAG - tag vector as a control or FLAG - Nup50 N - terminal fragment ( aa 1 - 120 of human ) . 24 h post transfection were lysed , FLAG - tagged immuno - isolated analyzed by western blotting ( B ) , with 10 % of inputs loaded . of western blot ( B , lower panel ) shows mean signal normalized on input of at least two independent experiments . Data points from experiments are indicated .

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HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1-120 of the human sequence). 24 h post transfection cells were lysed, FLAG-tagged proteins immuno-isolated and analyzed by western blotting (B), with 10% of the inputs loaded. The quantification of the western blot (B, lower panel) shows the mean signal intensity normalized on the input of at least two independent experiments. Data points from individual experiments are indicated.


( E ) 2 µM recombinant Ran , loaded with MANT - GDP was incubated with 2 mM GppNHp in buffer control , supplemented with 2 nM recombinant RCC1 , 20 nM recombinant Xenopus Nup50 , or RCC1 Nup50 together . GDP to GppNHp exchanged was monitored by decrease in MANT of liberated GDP - MANT . lower panel shows rate constant of each experimental condition with n = 4 independent experiment per condition , bars represent mean , data points are indicated .

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(E) 2 µM recombinant Ran, loaded with MANT-GDP was incubated with 2 mM GppNHp in buffer control, supplemented with 2 nM recombinant RCC1, 20 nM recombinant Xenopus Nup50 proteins, or RCC1 and Nup50 together. GDP to GppNHp exchanged was monitored by the decrease in MANT fluorescence of the liberated GDP-MANT. The lower panel shows the rate constant of each experimental condition with n=4 independent experiment per condition, bars represent the mean, individual data points are indicated.


( G ) Confocal microscopy of nuclei assembled for 120 min in mock depleted , Nup50 depleted ( ∆ Nup50 ) , Nup50 depleted Xenopus egg extracts supplemented with recombinant Xenopus wild - type Nup50 or RCC1 binding mutants . Nuclei were fixed in 4 % PFA 0 . 5 % glutaraldehyde , stained for NPCs ( mAB414 ) ( DAPI ) . Scale bar : 10 µm . Quantitation shows average of mAB414 positive nuclei for 100 randomly chosen substrates in each of three independent experiments . data points are indicated ( H ) Confocal microscopy of nuclei assembled for 120 min in mock depleted , Nup50 depleted ( ∆ Nup50 ) , Nup50 depleted Xenopus egg extracts supplemented with RCC1 excess as indicated . Nuclei were fixed in 4 % PFA 0 . 5 % glutaraldehyde , stained for NPCs ( mAB414 ) ( DAPI ) . Scale bar : 10 µm . Quantitation shows average of mAB414 positive nuclei for 100 randomly chosen substrates in each of three independent experiments . data points are indicated .

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(G) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant Xenopus wild-type Nup50 or RCC1 binding mutants. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated (H) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with RCC1 excess as indicated. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated.


Comparison of structural preservation in cryosections picked up with sucrose ( a ) MU ( b ) . In isolated hepatocytes , autophagy was induced for 30 min in suspension buffer before fixation . ( a ) Cryosection picked up with 2 . 3 M sucrose labeled for SOD . Note presence of large , " empty " gaps between sequestered contents of AV surrounding cytoplasm . ( b ) A similar section as a , but picked up with an MU mixture . AV are no longer distorted overall membranous structures are sharply delineated . SOD - labeling density of AVi is as that of cytosol , whereas AVd show much more intense labeling . ( Inset ) An AVi / d that is distinct from AVi by its single limiting . highly pleiomorphic AVd are also bound by a distinctive single , but in contrast to AVi / d , their contents are characterized by debris of digested material . Bars , 250 nm .

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Comparison of structural preservation in cryosections picked up with sucrose (a) and MU (b). In isolated hepatocytes, autophagy was induced for 30 min in suspension buffer before fixation. (a) Cryosection picked up with 2.3 M sucrose and labeled for SOD. Note the presence of large, "empty" gaps between sequestered contents of the AV and the surrounding cytoplasm. (b) A similar section as a, but picked up with an MU mixture. The AV are no longer distorted and the overall membranous structures are sharply delineated. The SOD-labeling density of AVi is as that of the cytosol, whereas AVd show much more intense labeling. (Inset) An AVi/d that is distinct from AVi by its single limiting membrane. The highly pleiomorphic AVd are also bound by a distinctive single membrane, but in contrast to AVi/d, their contents are characterized by debris of digested material. Bars, 250 nm.


Labeling patterns of cathepsin D CAIII in MU picked - up cryosections . ( a ) treated as in Fig . 1 b , but section was immunolabeled for cathepsin D , which is not detectable in AVi , moderately present in AVi / d , abundant in AVd . in b c had endocytosed 5 - nm gold tracer for 20 40 min , respectively , before fixation CAIII labeling . ( b ) fusion profile of a CAIII - positive AVi , an AVd which is without CAIII labeling , a VE that contains an endocytosed gold probe ( arrow ) . Close to AVd is another tracer - bearing VE . ( c ) Fusion profile of a tracer - bearing MVE an AVi / d that exhibits a single limiting a CAIII - labeling similar to that of cytoplasm . Bars , 200 nm .

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Labeling patterns of cathepsin D and CAIII in MU picked-up cryosections. (a) Cells treated as in Fig. 1 b, but the section was immunolabeled for cathepsin D, which is not detectable in the AVi, moderately present in the AVi/d, and abundant in AVd. Cells in b and c had endocytosed 5-nm gold tracer for 20 and 40 min, respectively, before fixation and CAIII labeling. (b) Complex fusion profile of a CAIII-positive AVi, an AVd which is without CAIII labeling, and a VE that contains an endocytosed gold probe (arrow). Close to the AVd is another tracer-bearing VE. (c) Fusion profile of a tracer-bearing MVE and an AVi/d that exhibits a single limiting membrane and a CAIII-labeling concentration similar to that of the cytoplasm. Bars, 200 nm.


Appearance of tracer in AV . were allowed to endocytose tracer particles continuously for designated periods of time . At each time , random sections were searched for AV + , AV + / 500 profiles were plotted . number of profiles examined ( denominator ) respective number of AV + scored ( numerator ) are listed above graph .

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Appearance of tracer in AV. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+, and AV+/500 cell profiles were plotted. The number of cell profiles examined (denominator) and the respective number of AV+ scored (numerator) are listed above the graph.


Tracer distribution in AV + types . were allowed to endocytose tracer particles continuously for designated periods of time . At each time , random sections were searched for AV + . results are categorized according to AV types plotted for relative distribution of tracers . total tracer are given on top of each bar . number in each block represents actual number of AV profiles , from which tracer are scored .

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Tracer distribution in AV+ types. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+. The results are categorized according to AV types and plotted for the relative distribution of tracers. The total tracer counts are given on top of each bar. The number in each block represents the actual number of AV profiles, from which tracer counts are scored.


Fusion profiles . exposed to 5 - nm gold tracers for 20 ( a c ) 25 min ( b ) before fixation . Sections were picked up by MU air - dried ( c ) or underwent immunolabeling with anti - SOD antibodies ( a b ) . ( a ) Fusion between a small VE ( arrow ) an AVi . A cluster of two gold particles ( arrowhead ) is also seen at another location . ( b ) Fusion between an electron lucent MVE ( arrow ) an AVd . ( c ) Fusion between an MVE ( arrow ) an AVd . Bars , 200 nm .

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Fusion profiles. Cells exposed to 5-nm gold tracers for 20 (a and c) and 25 min (b) before fixation. Sections were picked up by MU and air-dried (c) or underwent immunolabeling with anti-SOD antibodies (a and b). (a) Fusion between a small VE (arrow) and an AVi. A cluster of two gold particles (arrowhead) is also seen at another location. (b) Fusion between an electron lucent MVE (arrow) and an AVd. (c) Fusion between an MVE (arrow) and an AVd. Bars, 200 nm.


Endosome types involved in FP + . FP + were classified according to MVE VE involved . actual numbers of FP + analyzed are given above bars .

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Endosome types involved in FP+. The FP+ were classified according to MVE and VE involved. The actual numbers of FP+ analyzed are given above the bars.


ASGP - R labeling in FP + endosomes . exposed for 40 min to 5 - nm gold tracer . Section immunolabeled for ASGP - R with 10 - nm gold . ( a ) Low ASGP - R labeling in tracer - carrying endocytic vesicles ( arrowheads ) , adjacent to ( top ) or fusing with ( bottom ) an AVi / d . ( b ) ASGP - R labeling at plasma in endosomal elements near surface ( ex , extra space ) . Vesicles of MVE ( asterisk ) VE ( arrowhead ) type , containing endocytosed gold , are present in same . ( c ) As b . A VE - like vesicle with endocytosed gold low ASGP - R labeling ( arrowhead ) is shown connected to an ASGP - R rich element ( arrow ) , possibly a recycling endosome . Bars , 200 nm .

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ASGP-R labeling in FP+ and endosomes. Cells exposed for 40 min to 5-nm gold tracer. Section immunolabeled for ASGP-R with 10-nm gold. (a) Low ASGP-R labeling in tracer-carrying endocytic vesicles (arrowheads), adjacent to (top) or fusing with (bottom) an AVi/d. (b) ASGP-R labeling at the plasma membrane and in endosomal elements near the cell surface (ex, extra cellular space). Vesicles of the MVE (asterisk) and VE (arrowhead) type, containing endocytosed gold, are present in the same area. (c) As b. A VE-like vesicle with endocytosed gold and low ASGP-R labeling (arrowhead) is shown connected to an ASGP-R rich element (arrow), possibly a recycling endosome. Bars, 200 nm.


AV types involved in FP + . same FP + data as used in Fig . 6 were classified according to AV types .

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AV types involved in FP+. The same FP+ data as used in Fig. 6 were classified according to the AV types.


( A ) HeLa were exposed to 5 Gy ionizing radiation ( γIR ) , collected at indicated time points stained for RASSF1A γH2AX . for representative intra - RASSF1A foci distribution are shown . Nucleolar boundaries are marked with dashed lines .

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(A) HeLa cells were exposed to 5 Gy ionizing radiation (γIR), collected at the indicated time points and stained for RASSF1A and γH2AX. Images for representative intra-nuclear RASSF1A foci distribution are shown. Nucleolar boundaries are marked with dashed lines.


( B ) of number of with RASSF1A foci at time points presented in ( A ) . Error bars represent standard deviation derive from 3 independent experiments .

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(B) Quantification of the number of cells with RASSF1A foci at the time points presented in (A). Error bars represent standard deviation and derive from 3 independent experiments.


( C ) HeLa were treated with 5 Gy γIR 2 h post treatment fixed stained with indicated antibodies . ( D ) HeLa were irradiated with 5 Gy 2 h post treatment stained for RASSF1A ( R1A ) NUCLEOLIN ( NCL ) . ( E ) HeLa were treated with 50 ng / ml NCS for 30 min , washed 2 h later fixed stained with indicated antibodies . of RASSF1A + / γH2AX + foci or RASSF1A - / γH2AX + foci to closest nucleolus ( on Fibrillarin ( Fb ) ) was measured . Middle represents median boxes 25th 75th percentiles . whiskers mark smallest largest values .

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(C) HeLa cells were treated with 5 Gy γIR and 2 h post treatment fixed and stained with the indicated antibodies. (D) HeLa cells were irradiated with 5 Gy and 2 h post treatment stained for RASSF1A (R1A) and NUCLEOLIN (NCL). (E) HeLa cells were treated with 50 ng/ml NCS for 30 min, washed and 2 h later fixed and stained with the indicated antibodies. The distance of RASSF1A+/γH2AX+ foci or RASSF1A-/γH2AX+ foci to the closest nucleolus (based on Fibrillarin (Fb) staining) was measured. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values.


( F ) AsiSI expression in U2OS was induced by OHT were stained for RASSF1A γH2AX . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines . ( G ) of number of with RASSF1A foci in ( F ) . Error bars represent standard deviation derive from 3 independent experiments .

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(F) AsiSI expression in U2OS cells was induced by OHT and cells were stained for RASSF1A and γH2AX. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (G) Quantification of the number of cells with RASSF1A foci in (F). Error bars represent standard deviation and derive from 3 independent experiments.


( H ) U2OS with stable integration of a Lac operator ( LacO ) adjacent to an I - SceI endonuclease site were transfected with Lac Repressor ( LacR ) - mCherry GFP or GFP - NLS - HA - I - SceI . 24 h post transfection were fixed stained for 53BP1 or RASSF1A . ( I ) of LacO arrays enriched for 53BP1 or RASSF1A in ( H ) . Error bars represent standard deviation derive from 3 independent experiments .

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(H) U2OS cells with stable integration of a Lac operator (LacO) sequence adjacent to an I-SceI endonuclease site were transfected with Lac Repressor (LacR)-mCherry and GFP or GFP-NLS-HA-I-SceI. 24 h post transfection cells were fixed and stained for 53BP1 or RASSF1A. (I) Quantification of the LacO arrays enriched for 53BP1 or RASSF1A in (H). Error bars represent standard deviation and derive from 3 independent experiments.


( J ) HeLa were transfected with I - PpoI endonuclease stained for RASSF1A γH2AX . profiles of RASSF1A ( green ) γH2AX ( red ) signals across HeLa nuclei are shown . Position of scan indicated by yellow .

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(J) HeLa cells were transfected with I-PpoI endonuclease and stained for RASSF1A and γH2AX. Fluorescence intensity profiles of RASSF1A (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( A ) HeLa were transfected with I - PpoI mRNA collected at indicated time points . were co - stained for RASSF1A ( R1A ) NBS1 . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines . ( B ) of ( A ) . Error bars represent standard deviation derive from 3 independent experiments .

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(A) HeLa cells were transfected with I-PpoI mRNA and collected at the indicated time points. Cells were co-stained for RASSF1A (R1A) and NBS1. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (B) Quantification of (A). Error bars represent standard deviation and derive from 3 independent experiments.


( C ) Assessment of RASSF1A localisation after induction of rDNA DSBs using I - PpoI endonuclease in methanol fixed HeLa . profile of RASSF1A ( green ) LAMIN A / C ( red ) signals across HeLa nuclei in presence absence of rDNA DSBs in control I - PpoI treated . Position of scan indicated by yellow .

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

(C) Assessment of RASSF1A localisation after induction of rDNA DSBs using the I-PpoI endonuclease in methanol fixed HeLa cells. Fluorescence intensity profile of RASSF1A (green) and LAMIN A/C (red) signals across the HeLa nuclei in the presence and absence of rDNA DSBs in control and I-PpoI treated cells. Position of line scan indicated by the yellow line.


( D ) HeLa were transfected with I - PpoI mRNA 6 h later stained for RASSF1A - pS131 ( R1A - pS131 ) γH2AX . profile of RASSF1A - pS131 ( green ) γH2AX ( red ) signals across HeLa nuclei are shown . Position of scan indicated by yellow .

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(D) HeLa cells were transfected with the I-PpoI mRNA and 6 h later stained for RASSF1A-pS131 (R1A-pS131) and γH2AX. Fluorescence intensity profile of RASSF1A-pS131 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( E ) of with RASSF1A - pS131 positive nucleolar caps 6 h post I - PpoI transfection . Error bars represent standard deviation derive from 3 independent experiments . Representative are shown in Fig EV3A .

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(E) Quantification of cells with RASSF1A-pS131 positive nucleolar caps 6 h post I-PpoI transfection. Error bars represent standard deviation and derive from 3 independent experiments. Representative images are shown in Fig EV3A.


( F ) HeLa were treated with ATMi or ATRi prior to transfection with I - PpoI mRNA . 6 h post mRNA transfection were stained for RASSF1A - pS131 ( R1A - pS131 ) . ( G ) of ( F ) . Error bars represent standard deviation derive from 3 independent experiments .

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(F) HeLa cells were treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. 6 h post mRNA transfection cells were stained for RASSF1A-pS131 (R1A-pS131). (G) Quantification of (F). Error bars represent standard deviation and derive from 3 independent experiments.


( H ) HeLa were treated or not with ATM inhibitor ( ATMi ) followed by I - PpoI mRNA transfection 6 h later lysates were analyzed by western blot for indicated antibodies .

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(H) HeLa cells were treated or not with ATM inhibitor (ATMi) followed by I-PpoI mRNA transfection and 6 h later cell lysates were analyzed by western blot for the indicated antibodies.


( I ) HeLa were treated or not with ATMi followed by I - PpoI mRNA transfection 6 h later were fixed RASSF1A recruitment at rDNA DSBs was assessed . ( J ) of ( I ) . Error bars represent standard deviation derive from 3 independent experiments .

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(I) HeLa cells were treated or not with ATMi followed by I-PpoI mRNA transfection and 6 h later cells were fixed and RASSF1A recruitment at rDNA DSBs was assessed. (J) Quantification of (I). Error bars represent standard deviation and derive from 3 independent experiments.


( A ) HeLa with I - PpoI induced rDNA breaks were stained with indicated antibodies . Intra - nucleolar cap localization for RASSF1A 53BP1 in correlation with other was accessed . Boxed areas are shown in higher magnification .

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(A) HeLa cells with I-PpoI induced rDNA breaks were stained with the indicated antibodies. Intra-nucleolar cap localization for RASSF1A and 53BP1 in correlation with other proteins was accessed. Boxed areas are shown in higher magnification.


( B ) HeLa were transfected with I - PpoI mRNA stained for RASSF1A 53BP1 . profiles of RASSF1A ( green ) 53BP1 ( red ) signals across HeLa nuclei are shown . Position of scan indicated by yellow .

(B) HeLa cells were transfected with I-PpoI mRNA and stained for RASSF1A and 53BP1. Fluorescence intensity profiles of RASSF1A (green) and 53BP1 (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( C ) HeLa were treated with siRNAs against LUCIFERASE ( siLUC ) or 53BP1 ( si53BP1 ) , 48 h later transfected with I - PpoI mRNA , fixed stained for RASSF1A ( R1A ) γH2AX . ( D ) of ( C ) western blot from siLUCIFERASE ( siLUC ) or si53BP1 treated for indicated antibodies . Error bars represent standard deviation derive from 3 independent experiments .

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(C) HeLa cells were treated with siRNAs against LUCIFERASE (siLUC) or 53BP1 (si53BP1), 48 h later transfected with I-PpoI mRNA, fixed and stained for RASSF1A (R1A) and γH2AX. (D) Quantification of (C) and western blot analysis from siLUCIFERASE (siLUC) or si53BP1 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.


( E ) HeLa were treated with siRNAs against LUSIFERASE ( siLUC ) or RNF8 ( siRNF8 ) , 48 h later transfected for I - PpoI mRNA , fixed stained for RASSF1A γH2AX . ( F ) of ( E ) western blot from siLUCIFERASE ( siLUC ) or siRNF8 treated for indicated antibodies . Error bars represent standard deviation derive from 3 independent experiments .

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(E) HeLa cells were treated with siRNAs against LUSIFERASE (siLUC) or RNF8 (siRNF8), 48 h later transfected for I-PpoI mRNA, fixed and stained for RASSF1A and γH2AX. (F) Quantification of (E) and western blot analysis from siLUCIFERASE (siLUC) or siRNF8 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.


( G ) HeLa were treated with siRNA against 53BP1 , or an ATM inhibitor ( ATMi ) followed by 5 Gy of ionizing radiation ( γIR ) . were fixed 2 h post treatment stained for RASSF1A . Nucleolar boundaries are marked with dashed lines . ( H ) of with RASSF1A foci in ( G ) . Error bars represent standard deviation derive from 3 independent experiments .

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(G) HeLa cells were treated with siRNA against 53BP1, or an ATM inhibitor (ATMi) followed by 5 Gy of ionizing radiation (γIR). Cells were fixed 2 h post treatment and stained for RASSF1A. Nucleolar boundaries are marked with dashed lines. (H) Quantification of cells with RASSF1A foci in (G). Error bars represent standard deviation and derive from 3 independent experiments.


( I ) Western blot from total extracts with indicated treatments RASSF1A immuneprecipitates ( IPs ) with indicated antibodies . IgG immunoprecipitation served as a control .

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(I) Western blot analysis from total cell extracts with the indicated treatments and RASSF1A immuneprecipitates (IPs) with the indicated antibodies. IgG immunoprecipitation served as a control.


( J ) Western blot from total extracts RASSF1A immunoprecipitates with indicated treatments with indicated antibodies . IgG immunoprecipitation served as a control .

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(J) Western blot analysis from total cell extracts and RASSF1A immunoprecipitates with the indicated treatments with the indicated antibodies. IgG immunoprecipitation served as a control.


( K ) U2OS were transfected with FLAG - RASSF1A ( FLAG - R1A ) , FLAG - RASSF1A131A ( FLAG - R1A131A ) or pcDNA3 24 h later transfected with I - PpoI mRNA . lysates were subjected to immunoprecipitation against FLAG . Western blot of total extracts IPs is shown .

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(K) U2OS cells were transfected with FLAG-RASSF1A (FLAG-R1A), FLAG-RASSF1A131A (FLAG-R1A131A) or pcDNA3 and 24 h later transfected with the I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation against FLAG. Western blot analysis of total cell extracts and the IPs is shown.


( A ) U2OS were transfected with either HA - 53BP1WT ( aa1 - 1972 ) , HA - 53BP1ΔBRCT ( aa 1 - 1709 ) , HA - 53BP1ΔΝterminus ( aa 921 - 1972 ) or empty pcDNA3 vector 24 h later transfected with I - PpoI mRNA . lysates were subjected to immunoprecipitation with an HA tag antibody . Western blot of total extracts IPs is shown .

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(A) U2OS cells were transfected with either HA-53BP1WT (aa1-1972), HA-53BP1ΔBRCT (aa 1-1709), HA-53BP1ΔΝterminus (aa 921-1972) or empty pcDNA3 vector and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with an HA tag antibody. Western blot analysis of total cell extracts and the IPs is shown.


( B ) U2OS were transfected with either full MYC - RASSF1A or with indicated RASSF1A deletion mutants 24 h later transfected with I - PpoI mRNA . lysates were subjected to immunoprecipitation with a MYC - tag antibody . Western blot of total extracts IPs is shown .

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(B) U2OS cells were transfected with either full length MYC-RASSF1A or with the indicated RASSF1A deletion mutants and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with a MYC-tag antibody. Western blot analysis of the total cell extracts and the IPs is shown.


( C ) HeLa were treated with siLUCIFERASE ( siLUC ) or si53BP1 48 h later transfected with I - PpoI mRNA stained for ATM - pS1981 . ( D ) of ( C ) . Error bars represent standard deviation derive from 3 independent experiments .

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(C) HeLa cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for ATM-pS1981. (D) Quantification of (C). Error bars represent standard deviation and derive from 3 independent experiments.


( E - H ) HeLa were treated with siLUCIFERASE ( siLUC ) or two different siRNAs against RASSF1A ( siR1A _ 1 siR1A _ 2 ) 48 h later were transfected with I - PpoI mRNA to induce rDNA DSBs . 6 h post mRNA transfection , were stained for V5 to identify I - PpoI transfected other indicated antibodies . Representative ( E , F ) of ( G , H ) for indicated antibodies are shown . Error bars represent standard deviation derive from 3 independent experiments .

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

(E-H) HeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2) and 48 h later cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (E, F) and quantification of staining (G, H) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.


( I ) HeLa were treated with siLUCIFERASE ( siLUC ) , siRASSF1A or si53BP1 48 h later transfected with I - PpoI mRNA . were fixed underwent ImmunoFISH against UBF an rDNA probe . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines . ( J ) HeLa were treated with siLUCIFERASE ( siLUC ) , siRASSF1A or si53BP1 48 h later transfected with I - PpoI mRNA . were fixed underwent ImmunoFISH against ATM - pS1981 an rDNA probe . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines .

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

(I) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against UBF and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (J) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against ATM-pS1981 and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines.


( K ) HeLa were treated with siLUCIFERASE ( siLUC ) or siRASSF1A followed by I - PpoI mRNA transfection . 6 h later were stained for 53BP1 . ( L ) of ( K ) . Error bars represent standard deviation derive from 3 independent experiments .

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(K) HeLa cells were treated with siLUCIFERASE (siLUC) or siRASSF1A followed by I-PpoI mRNA transfection. 6 h later cells were stained for 53BP1. (L) Quantification of (K). Error bars represent standard deviation and derive from 3 independent experiments.


( M ) U2OS were transfected with MYC - RASSF1A ( 1 - 340 ) or a RASSF1A mutant that lacks SARAH domain , MYC - RASSF1A ( 1 - 288 ) . were transfected with I - PpoI mRNA stained with indicated antibodies .

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(M) U2OS cells were transfected with MYC-RASSF1A (1-340) or a RASSF1A mutant that lacks the SARAH domain, MYC-RASSF1A (1-288). Cells were transfected with I-PpoI mRNA and stained with the indicated antibodies.


( N ) U2OS were transfected with MYC - RASSF1A ( 1 - 340 ) or MYC - RASSF1A ( 1 - 288 ) . 24 h later were transfected with I - PpoI mRNA harvested after 6 h . lysates underwent fractionation bound fraction was analyzed by western blot .

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(N) U2OS cells were transfected with MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and harvested after 6 h. Cell lysates underwent cell fractionation and the chromatin bound fraction was analyzed by western blot.


( O ) U2OS were treated with siRNA against RASSF1A followed by transfection with MYC - tag , MYC - RASSF1A ( 1 - 340 ) or MYC - RASSF1A ( 1 - 288 ) . 24 h later were transfected with I - PpoI mRNA stained for MYC expression ATM - pS1981 .

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(O) U2OS cells were treated with siRNA against RASSF1A followed by transfection with MYC-tag, MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and stained for MYC expression and ATM-pS1981.


( P ) of with ATM - pS1981 positive nucleolar caps in ( O ) . Error bars represent standard deviation derive from 3 independent experiments .

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(P) Quantification of cells with ATM-pS1981 positive nucleolar caps in (O). Error bars represent standard deviation and derive from 3 independent experiments.


( A ) HeLa were pre - treated with ATMi or ATRi prior to transfection with I - PpoI mRNA . were stained for RPA quantified . Error bars represent standard deviation derive from 3 independent experiments . ( B ) HeLa were pre - treated with ATMi or ATRi prior to transfection with I - PpoI mRNA . were stained for RAD51 quantified . Error bars represent standard deviation derive from 3 independent experiments .

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(A) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RPA and quantified. Error bars represent standard deviation and derive from 3 independent experiments. (B) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RAD51 and quantified. Error bars represent standard deviation and derive from 3 independent experiments.


( C - F ) HeLa were treated with siLUCIFERASE ( siLUC ) or two different siRNAs against RASSF1A ( siR1A _ 1 siR1A _ 2 ) 48 h later were transfected with I - PpoI mRNA to induce rDNA DSBs . 6 h post mRNA transfection , were stained for V5 to identify I - PpoI transfected other indicated antibodies . Representative ( C , E ) of ( D , F ) for indicated antibodies are shown . Error bars represent standard deviation derive from 3 independent experiments .

(C-F) HeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2) and 48 h later cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (C, E) and quantification of staining (D, F) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.


( G ) Western blot from HeLa extracts treated with indicated siRNAs for indicated antibodies .

(G) Western blot analysis from HeLa cell extracts treated with the indicated siRNAs for the indicated antibodies.


( H ) were treated with siLUCIFERASE ( siLUC ) or si53BP1 48 h later transfected with I - PpoI mRNA stained for RPA . ( I ) of ( H ) . 107 values were analysed in siLUC 90 values were analysed in si53BP1 .

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(H) Cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for RPA. (I) Quantification of (H). 107 values were analysed in siLUC and 90 values were analysed in si53BP1.


( J ) U2OS were treated with siLUCIFERASE ( siLUC ) , siRASSF1A ( siR1A ) or si53BP1 , treated with 10 μM BrdU for 24 h prior to I - PpoI mRNA transfection . 6 h post I - PpoI treated were pre - extracted , fixed stained for BrdU under non denaturing conditions to visualize ssDNA . ( K ) of ( J ) . Fold change of BrdU signal relative to siLUC is shown . Error bars represent standard deviation derive from 3 independent experiments .

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

(J) U2OS cells were treated with siLUCIFERASE (siLUC), siRASSF1A (siR1A) or si53BP1, treated with 10 μM BrdU for 24 h prior to I-PpoI mRNA transfection. 6 h post I-PpoI treated cells were pre-extracted, fixed and stained for BrdU under non denaturing conditions to visualize ssDNA. (K) Quantification of (J). Fold change of nuclear BrdU signal relative to siLUC is shown. Error bars represent standard deviation and derive from 3 independent experiments.


( L ) HeLa were transfected with I - PpoI endonuclease stained for MST2 γH2AX . profiles of MST2 ( green ) γH2AX ( red ) signals across HeLa nuclei are shown . Position of scan indicated by yellow .

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(L) HeLa cells were transfected with I-PpoI endonuclease and stained for MST2 and γH2AX. Fluorescence intensity profiles of MST2 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( M ) Validation of MST2 siRNA with western blot .

(M) Validation of the MST2 siRNA with western blot.


( N ) HeLa treated with siLUCIFERASE ( siLUC ) or siMST2 48 h later transfected with I - PpoI mRNA . were treated with 5 - EU for 30 min prior to fixation assessed for incorporation . ( O ) of 5 - EU in siLUCIFERASE ( siLUC ) or siMST2 treated transfected with I - PpoI mRNA . Middle represents median boxes 25th 75th percentiles . whiskers mark smallest largest values . 100 values were analyzed in each condition .

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(N) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were treated with 5-EU for 30 min prior to fixation and assessed for incorporation. (O) Quantification of 5-EU intensity in siLUCIFERASE (siLUC) or siMST2 treated cells transfected with I-PpoI mRNA. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values. 100 values were analyzed in each condition.


( P ) HeLa were treated with siLUCIFERASE ( siLUC ) or siMST2 48 h post transfection were transfected with I - PpoI mRNA . 6 h post transfection were fixed stained for RASSF1A ( R1A ) γH2AX . ( Q ) of ( P ) . Error bars represent standard deviation derive from 3 independent experiments .

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(P) HeLa cells were treated with siLUCIFERASE (siLUC) or siMST2 and 48 h post transfection cells were transfected with I-PpoI mRNA. 6 h post transfection cells were fixed and stained for RASSF1A (R1A) and γH2AX. (Q) Quantification of (P). Error bars represent standard deviation and derive from 3 independent experiments.


( R - W ) HeLa treated with siLUCIFERASE ( siLUC ) or siMST2 48 h later transfected with I - PpoI mRNA . were stained for ATM - pS1981 ( R ) , KAP1 - pS824 ( T ) RPA ( V ) quantified ( S , U , W ) . 108 values in ( S ) , 95 values in ( U ) 100 values in ( W ) were analysed .

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(R-W) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were stained for ATM-pS1981 (R), KAP1-pS824 (T) and RPA (V) and quantified (S, U, W). 108 values in (S), 95 values in (U) and 100 values in (W) were analysed.


( Β ) Clonogenic survival of HeLa treated with indicated siRNAs indicated doses of Ionizing radiation ( γIR ) . Survival fraction was calculated . Error bars represent derive from 3 independent experiments .

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(Β) Clonogenic survival analysis of HeLa cells treated with the indicated siRNAs and the indicated doses of Ionizing radiation (γIR). Survival fraction was calculated. Error bars represent SEM and derive from 3 independent experiments.


( C ) Clonogenic survival representative of HeLa treated with indicated siRNAs followed by I ‐ PpoI WT or I - PpoI H98A mRNA transfections . Survival I - PpoI WT / I - PpoI H98A in each siRNA condition is presented . Error bars represent standard deviation derive from 3 independent experiments .

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(C) Clonogenic survival analysis and representative images of HeLa cells treated with the indicated siRNAs followed by I‐PpoI WT or I-PpoI H98A mRNA transfections. Survival ratio I-PpoI WT/I-PpoI H98A in each siRNA condition is presented. Error bars represent standard deviation and derive from 3 independent experiments.


( E ) 45S rDNA copy number in ' tumors compared to adjacent controls in LUAD cohort . 67 samples 43 samples from adjacent tissue were analyzed . ( F ) 45S rDNA copy number in LUAD samples on RASSF1A CpG promoter methylation . 67 samples were analysed .

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(E) 45S rDNA copy number analysis in patients' tumors compared to adjacent controls in the LUAD cohort. 67 tumor samples and 43 samples from adjacent tissue were analyzed. (F) 45S rDNA copy number analysis in the LUAD patient samples based on RASSF1A CpG promoter methylation. 67 samples were analysed.


IE were isolated binding to HBMEC HDMEC was determined under flow conditions . Number of IE bound per mm2 EC surface was calculated shown is mean ± 95 % CI for 26 CM 33 UM cases on HBMEC 21 CM 35 UM cases on HDMEC on a log scale . P - value was calculated by two - unpaired t - . dotted is 20 IE / mm2 , cut - off value for inclusion of inhibition data .

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IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions. Number of IE bound per mm2 EC surface was calculated and shown is the mean ± 95% CI for 26 CM and 33 UM cases on HBMEC and 21 CM and 35 UM cases on HDMEC on a log scale. P-value was calculated by two-tailed unpaired t-test. The dotted line is 20 IE/mm2, the cut-off value for inclusion of the inhibition data.


IE were isolated binding to HBMEC HDMEC was determined under flow conditions in absence presence of 5 µg / ml αICAM - 1 or αCD36 antibody or 50 µg / ml rEPCR . Number of IE bound per mm2 EC surface was determined .

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IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 µg/ml αICAM-1 or αCD36 antibody or 50 µg/ml rEPCR. Number of IE bound per mm2 EC surface was determined.


IE were isolated binding to HBMEC HDMEC was determined under flow conditions in absence presence of 5 µg / ml αICAM - 1 or αCD36 antibody or 50 µg / ml rEPCR . Number of IE bound per mm2 EC surface was determined . Using same data , inhibition by αICAM - 1 antibody was calculated relatively to binding in absence of antibody . Using same data , inhibition by rEPCR was calculated relatively to binding in absence of inhibitor . Using same data , inhibition by αCD36 antibody was calculated relatively to binding in absence of antibody .

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IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 µg/ml αICAM-1 or αCD36 antibody or 50 µg/ml rEPCR. Number of IE bound per mm2 EC surface was determined. Using the same data, percentage inhibition by αICAM-1 antibody was calculated relatively to binding in the absence of antibody. Using the same data, percentage inhibition by rEPCR was calculated relatively to binding in the absence of inhibitor. Using the same data, percentage inhibition by αCD36 antibody was calculated relatively to binding in the absence of antibody.


( A ) Ventricular to tibia ( VW / TL ) of littermate control ( LC ) or cardiomyocyte - specific NCoR1 knockout ( CMNKO ) mice . n = 10 : 9 : 12 : 7 .

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(A) Ventricular weight to tibia length ratio (VW/TL) of littermate control (LC) or cardiomyocyte-specific NCoR1 knockout (CMNKO) mice. n=10:9:12:7.


( B ) Ventricular to ( VW / BW ) of LC or CMNKO mice . n = 10 : 8 : 12 : 7 .

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(B) Ventricular weight to body weight ratio (VW/BW) of LC or CMNKO mice. n=10:8:12:7.


( C ) Representative H & E of cross sections of left ventricles . Scale bar : 50μm .

(C) Representative H&E staining of cross sections of left ventricles. Scale bar: 50μm.


( D ) Representative wheat germ agglutinin ( WGA ) of cross section of left ventricles . Scale bar : 10μm .

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(D) Representative wheat germ agglutinin (WGA) staining of cross section of left ventricles. Scale bar: 10μm.


( E ) of cardiomyocyte . n = 11 : 10 : 8 : 8 .

(E) Quantification of cardiomyocyte size. n=11:10:8:8.


( F ) qRT - PCR of hypertrophy - related genes in left ventricles . n = 9 : 9 : 11 : 8 .

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(F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=9:9:11:8.


( G ) Ejection fraction measured by echocardiography . n = 9 : 7 : 14 : 10 . Student ' s t was used for statistical .

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(G) Ejection fraction measured by echocardiography. n=9:7:14:10. Student's t test was used for statistical analysis.


( H ) Fractional shortening measured by echocardiography . n = 9 : 7 : 14 : 10 . Student ' s t was used for statistical .

(H) Fractional shortening measured by echocardiography. n=9:7:14:10. Student's t test was used for statistical analysis.


( A ) Representative of hearts from LC or CMNKO mice subjected to sham operation or AAC for 2 . Scale bar : 0 . 5cm .

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(A) Representative images of hearts from LC or CMNKO mice subjected to sham operation or AAC for 2 weeks. Scale bar: 0.5cm.


( B ) VW / BW of LC or CMNKO mice 2 after sham operation or AAC . n = 9 : 9 : 16 : 15 .

(B) VW/BW of LC or CMNKO mice 2 weeks after sham operation or AAC. n=9:9:16:15.


( C ) Representative H & E of cross sections of left ventricles . Scale bar : 25μm .

(C) Representative H&E staining of cross sections of left ventricles. Scale bar: 25μm.


( D ) Representative WGA . Scale bar : 10μm .

(D) Representative WGA staining. Scale bar: 10μm.


( E ) of cardiomyocyte . n = 10 : 10 : 8 : 9 .

(E) Quantification of cardiomyocyte size. n=10:10:8:9.


( F ) qRT - PCR of hypertrophy - related genes in left ventricles . n = 8 : 7 : 15 : 15 .

(F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=8:7:15:15.


( G ) Representative picrosirius red of cross sections of left ventricles . Scale bar : 50μm .

(G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 50μm.


( H ) of fibrotic areas . n = 11 : 10 : 10 : 9 .

(H) Quantification of fibrotic areas. n=11:10:10:9.


( I ) qRT - PCR of fibrosis - related genes in left ventricles . n = 8 : 7 : 15 : 15 .

(I) qRT-PCR analysis of fibrosis-related genes in left ventricles. n=8:7:15:15.


( J ) Ejection fraction measured by echocardiography . n = 9 : 7 : 14 : 12 .

(J) Ejection fraction measured by echocardiography. n=9:7:14:12.


( K ) Fractional shortening measured by echocardiography . n = 9 : 7 : 14 : 12 .

(K) Fractional shortening measured by echocardiography. n=9:7:14:12.


( L ) Lung to ( LW / BW ) of LC or CMNKO mice 2 after sham operation or AAC . n = 8 : 7 : 16 : 15 .

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(L) Lung weight to body weight ratio (LW/BW) of LC or CMNKO mice 2 weeks after sham operation or AAC. n=8:7:16:15.


( M ) Cumulative survival rate of LC or CMNKO mice subjected to AAC . n = 12 : 10 . Two - way ANOVA followed by Bonferoni post - was used for statistical .

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(M) Cumulative survival rate of LC or CMNKO mice subjected to AAC. n=12:10. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( A ) Western blotting of NCoR1 in neonatal rat ventricular myocytes ( NRVMs ) transfected with siControl or siNCoR1 for 3 days . siControl indicates control siRNA ; siNCoR1 , NCoR1 siRNA .

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(A) Western blotting analysis of NCoR1 in neonatal rat ventricular myocytes (NRVMs) transfected with siControl or siNCoR1 for 3 days. siControl indicates control siRNA; siNCoR1, NCoR1 siRNA.


( B ) Representative immunofluorescence of α - Actinin in NRVMs transfected with siRNA for 48h then treated with vehicle ( H2O ) or phenylephrine ( PE ) for another 48h . Scale bar : 50μm .

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(B) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or phenylephrine (PE) for another 48h. Scale bar: 50μm.


( C ) of surface of α - Actinin - positive NRVMs with or without NCoR1 knockdown . A total of 40 NRVMs was randomly chosen from 4 replicate coverslips for each group used for statistical .

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(C) Quantification of the surface area of α-Actinin-positive NRVMs with or without NCoR1 knockdown. A total of 40 NRVMs was randomly chosen from 4 replicate coverslips for each group and used for statistical analysis.


( D ) qRT - PCR of Acta1 Nppa in NRVMs with or without NCoR1 knockdown . n = 4 : 4 .

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(D) qRT-PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 knockdown. n=4:4.


( E ) Western blotting of NCoR1 - flag in NRVMs infected by control lentivirus ( Control ) or NCoR1 - flag lentivirus ( NCoR1 - OV ) for 4 days .

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(E) Western blotting analysis of NCoR1-flag in NRVMs infected by control lentivirus (Control) or NCoR1-flag lentivirus (NCoR1-OV) for 4 days.


( F ) Representative immunofluorescence of α - Actinin in NRVMs infected with lentivirus for 48h then treated with vehicle or PE for another 48h . Scale bar : 50μm .

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(F) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm.


( G ) of surface of α - Actinin - positive NRVMs with or without NCoR1 overexpression . A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group used for statistical .

(G) Quantification of the surface area of α-Actinin-positive NRVMs with or without NCoR1 overexpression. A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( H ) qRT - PCR of Acta1 Nppa in NRVMs with or without NCoR1 overexpression . n = 4 : 3 . Data represent three independent experiments . Two - way ANOVA followed by Bonferoni post - was used for statistical .

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(H) qRT-PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 overexpression. n=4:3. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( A ) Representative immunofluorescence of α - Actinin in NRVMs transfected with siRNA for 48h then treated with vehicle ( H2O ) or PE for another 48h . Scale bar : 50μm . siMEF2a indicates MEF2a siRNA .

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(A) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or PE for another 48h. Scale bar: 50μm. siMEF2a indicates MEF2a siRNA.


( B ) of surface of α - Actinin - positive NRVMs with or without knockdown of NCoR1 / or MEF2a . A total of 15 - 20 NRVMs was randomly chosen from 3 replicate coverslips for each group used for statistical .

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(B) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2a. A total of 15-20 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( C ) Representative immunofluorescence of α - Actinin in NRVMs transfected with siRNA for 48h then treated with vehicle or PE for another 48h . Scale bar : 50μm . siMEF2d indicates MEF2d siRNA .

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(C) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm. siMEF2d indicates MEF2d siRNA.


( D ) of surface of α - Actinin - positive NRVMs with or without knockdown of NCoR1 / or MEF2d . A total of 15 - 19 NRVMs was randomly chosen from 3 replicate coverslips for each group used for statistical .

(D) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2d. A total of 15-19 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( E ) qRT - PCR of Acta1 Nppa in NRVMs . n = 4 . Data represent three independent experiments . Student ' s t was used for statistical .

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(E) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4. Data represent three independent experiments. Student's t test was used for statistical analysis.


( A ) Luciferase assays of Acta1 Nppa promoters in HEK293FT transfected with NCoR1 - Flag / or MEF2a - HA or with empty plasmids . Student ' s t was used for statistical

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(A) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with NCoR1-Flag and/or MEF2a-HA or with empty plasmids. Student's t test was used for statistical analysis


( B ) Co - immunoprecipitation ( Co - IP ) of NCoR1 MEF2a in HEK293FT transfected with full - NCoR1 - Flag MEF2a - HA .

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(B) Co-immunoprecipitation (Co-IP) analysis of NCoR1 and MEF2a in HEK293FT cells transfected with full-length NCoR1-Flag and MEF2a-HA.


( C ) Co - IP of NCoR1 truncated MEF2a in HEK293FT . Schematic illustration of MEF2a - HA construct is shown above Co - IP results .

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(C) Co-IP analysis of NCoR1 and truncated MEF2a in HEK293FT cells. Schematic illustration of MEF2a-HA construct is shown above the Co-IP results.


( D ) Co - IP of MEF2a truncated NCoR1 in HEK293FT . Schematic illustration of NCoR1 - Flag construct is shown above Co - IP results .

(D) Co-IP analysis of MEF2a and truncated NCoR1 in HEK293FT cells. Schematic illustration of NCoR1-Flag construct is shown above the Co-IP results.


( E ) Luciferase assays of Acta1 Nppa promoters in HEK293FT transfected with full - MEF2a domain - specific NCoR1 . Student ' s t was used for statistical

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(E) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with full-length MEF2a and domain-specific NCoR1. Student's t test was used for statistical analysis


( F ) Western blotting of NCoR1 - flag in NRVMs infected by control lentivirus ( Control ) or NCoR1 ( 1939 - 2453 ) - flag lentivirus [ NCoR1 ( 1939 - 2453 ) - OV ] for 4 days .

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(F) Western blotting analysis of NCoR1-flag in NRVMs infected by control lentivirus (Control) or NCoR1 (1939-2453)-flag lentivirus [NCoR1(1939-2453)-OV] for 4 days.


( G ) Representative immunofluorescence of α - Actinin in NRVMs infected with lentivirus for 48h then treated with vehicle ( DMSO ) or PE for another 48h . Scale bar : 50μm .

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(G) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle (DMSO) or PE for another 48h. Scale bar: 50μm.