Fig. 5: SURF2 depletion increases free 5S binding to MDM2 and increases cell sensitivity to nucleolar stress.

a Detection of proteins associated with RPL5-Flag. Cell extracts produced from U2OS cells overexpressing RPL5-FLAG or not (control) and differentially treated by actinomycin D addition for 24 h (at 10 ng/mL) or not (ACTD + or −) and transfected with scrambled siRNAs (siSCR) or directed against SURF2 (siSurf2) were used to perform immuno-precipitation on beads coated with anti-flag. 10% of the inputs were loaded (In) aside IPs (IP) on a gel to perform western blots using antibodies directed against the indicated proteins to analyze the co-purification efficiency of the different factors. b Quantification of the co-purification efficiency with RPL5-Flag observed (a) for the indicated proteins (n = 3). Results are represented as a comparison of the enrichments observed in cells treated with siRNAs directed against SURF2 (siSurf2) normalized to the ones observed in cells treated with scrambled siRNAs (siSCR). Two-tailed t-test analysis was used for statistics. Data are presented as mean values +/− SD. Significant differences are indicated by stars (p-value \(\le\)0.05*; \(\le\)0.01**;\(\,\le\)0.001*** and \(\le\)0.0001****) or with the precise p-value on the graph (p). c Quantification of the signals corresponding to the indicated proteins in the different inputs of western-blots analyses (a) from independent experiments n = 4 (original data are provided in the source data file). Two-tailed t test analysis was used for statistics. Data are presented as mean values +/− SD. Significant differences are indicated by stars (p-value \(\le\)0.05*; \(\le\)0.01**;\(\,\le\)0.001*** and \(\le\)0.0001****) or with the precise p-value on the graph (p). d DNA content analysis of U2OS cells treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2; ACTD: actinomycin D at 10 ng/mL for 24 h) by FACS. Quantification of U2OS cell repartition in the different phases of the cell cycle following different treatments (n = 3). Data are presented as mean values +/− SD. One-way Anova Test was used for statistics. Significant differences are indicated by stars (p-value \(\le\)0.05*; \(\le\)0.01**;\(\,\le\)0.001*** and \(\le\)0.0001****) or with the precise p-value on the graph (p). e, f Proliferation analysis of differentially treated U2OS cells. Cells are treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2; ACTD: actinomycin D at 10 ng/mL for 24 h). e Cells are platted on 6 well plate after being transfected with the indicated siRNAs, 24 h before being analyzed, cells were treated with actinomycin D (10 ng/mL) or H2O. Cells were stained with crystal violet to take a picture (n = 3) scale bar (0.5 mm). f Fixed crystal violet was resolubilized and quantified by absorbance for each condition after different time exposure to treatments (n = 3). g Cell apoptosis assays with an annexin-V-FITC and propidium iodide of differentially treated U2OS cells. Both apoptosis and necrosis are regrouped as dead cells (n = 3). Data are presented as mean values +/− SD. The One-way ANOVA test was used for statistics. Significant differences are indicated by stars (p-value \(\le\)0.05*; \(\le\)0.01**;\(\,\le\)0.001*** and \(\le\)0.0001****) or with the precise p-value on the graph (p). Source data are provided as a Source Data file.