Response of p53siRNA U2-OS cells to etoposide.

<p>(<b>A</b>) Inhibition of p53 expression in p53siRNA U2-OS. Actin was used as loading control. (<b>B</b>) p53siRNA U2-OS were less sensitive to etoposide when compared with parental and Ctrl U2-OS;). Student’s test from three independent experiments indicated significantly higher IC₅₀ mean values at 72 h of treatment in p53siRNA U2-OS than in Ctrl and parental U2-OS cells; p = 0.05 (<b>C</b>) Etoposide treatment did not induce mature miR-34a expression in p53siRNA U2-OS, as opposed to Ctrl U2-OS. (<b>D</b>) p53siRNA U2-OS cells presented CpG island methylation (M-MSP) of one of the two alleles of miR-34a. In Ctrl U2-OS both alleles were unmethylated. (<b>E</b>) p53siRNA transfection determined lengthening of G2/M phase after 48 h of etoposide treatment when compared to untreated cells. (<b>F</b>) Western blot of cyclin D1 and CDK4 in p53siRNA cell showed increased amount of CDK4 linked to cyclin D1 and total CDK4 after etoposide treatment when compared to control. No differences in cyclin D1 levels were seen. <i>Ctrl = siRNA negative control duplex; C = Untreated cells; T = Etoposide treated cells.</i>