Fig. 1: Silencing of SRSF3 caused p21cip1/waf1 upregulation and G2/M arrest in cellular senescence.
From: Splicing factor SRSF3 represses translation of p21cip1/waf1 mRNA
A U2OS cells were transiently transfected with either control siRNA or two different SRSF3 siRNAs and then subjected to cell cycle analysis by flow cytometry. B Flow cytometer histograms forward scatter (FSC) and side scatter (SSC) for siControl or siSRSF3 transfection in SW480 cells. C Representative picture of SA-β-Gal staining in SW480 cells. Senescence β-Galactosidase staining kit was used for the assay according to the manufacturer’s protocol. Scale bar, 100 μm. D Whole cell lysates from siControl or SRSF3-depleted SW480 cells were incubated with human cell stress array membranes. Each pair of the most positive protein dots is numbered with the identification of the corresponding protein listed. E Levels of indicated protein were quantified after normalized control. Error bars indicate mean ± SD of spots. F Associations between SRSF3, CDKN1A, NFkB1, p38-MAPK1, SIRT2, HIF1a, CDKN1B, and TP53 levels and tumor tissues were identified by analyzing the TCGA-Colon datasets. Positive correlations are expressed as mean Spearman r, and p values are for a two-tailed Student’s t-test. G Expression of SRSF3, CDKN1A, NFkB1, p38-MAPK1, SIRT2, HIF1a, CDKN1B, and TP53 mRNA in colon cancer from TCGA database. Data are shown as mean ± SD. ns not significant; ***P < 0.001, two-tailed Student’s t-test. H Expression of SRSF3 mRNA in 8 primary cancer types from TCGA database. Data are shown as mean ± SD. ns, not significant; ***P < 0.001, two-tailed Student’s t-test. I Expression of p21cip1/waf1 (CDKN1A) mRNA in 8 primary cancer types from TCGA database. Data are shown as mean ± SD. ns not significant; ***P < 0.001, two-tailed Student’s t-test. J SW480 cells were transiently transfected with either control siRNA or two different SRSF3 siRNAs, and the levels of endogenous p21cip1/waf1, SRSF2, and β-actin were analyzed by western blotting. K The siSRSF3-3′UTR targeting 3′UTR region of SRSF3 mRNA was co-transfected with Flag-SRSF3 plasmid DNA construct in SW480 cells. Western blot analysis with anti- p21cip1/waf1, anti-Flag, anti-SRSF3, and anti-β-actin antibodies.
