EWS downregulation affects apoptosis, cell cycle and proliferation.

<p><b>A</b>. Decrease in the relative mRNA levels of FBXW7 upon knocking down EWS. <b>B</b>. Relative mRNA levels of FUS, EWS and TAF15 (FET family proteins) upon EWS knock down. <b>C</b>. Total percentage of living, necrotic and apoptotic cells after EWS and scrambled siRNA knockdown are represented on the bars. Apoptotic cells are defined by the sum of population of cells in early apoptosis and late apoptosis. Mock, scrambled and EWS KD had 10%, 11.8% and 20% apoptotic cells and 17%, 22% and 30% of necrotic cells respectively. The P values refer to the apoptotic cell population. <b>D</b>. Rescue effect upon co-transfection of EWS siRNA and CCDC6 overexpression. Bars represent total percentage of living, apoptotic and necrotic cells. 50nM siRNA, 100ng of empty vector and 100ng of CCDC6 expression vector were transfected. Mock and scrambled had 15% and 18.6% of apoptotic cells, EWS KD and EWS KD+ Empty vector had 39% and 40.5% apoptotic cells respectively and EWS KD+ CCDC6 vector had only 24% apoptotic cells. The P values refer to the apoptotic cell population. <b>E</b>. Proliferation rates on three consecutive days using CCK8 assay was calculated by measuring the absorbance which is proportional to the amount of living cells. <b>F</b>. Quantification of cell cycle distribution. The bars indicate the % of cells in each cell cycle phase subG1, G0/G1, S and G2/M phase. Mock has 11.3%, 44.7%, 16%, 28% of cells in subG1, G0/G1, S and G2/M phase respectively. 16%, 42%, 15%, 27% of cells for scrambled and 37%, 44%, 5%, 14% of cells for EWS KD in each phase respectively. The calculated P values refer to the cells in S phase. Data in all the above figures (A-F) is presented as mean SEM values (*P<0.05; where n = 3 per group).