Figure 4

Effect of enforced overexpresson of XIAP IRES RNA on the expression of MDM2 and XIAP and on cancer cell growth. A, SK-N-SH cells were transfected for 24 h with the indicated amounts of pRNA-CMV3.1/Puro XIAP IRES RNA or pRNA-CMV3.1/Puro XIAP non-IRES RNA (control RNA) plasmids and the expression of MDM2, XIAP, MDM4 and p53 proteins was detected by Western blot. B, turnover of proteins as indicated in SK-N-SH cells transfected with XIAP IRES RNA or control RNA, as detected by pulse-chase assay. C, SK-N-SH cells stably transfected with either XIAP IRES RNA or control RNA expression plasmids were incubated in medium at an initial concentration of 10⁴/ml and then counted every day. Data shown: total number of cells (mean ± SD for triplicate cultures, *p < 0.05). D, clonogenic assay of MDM2 or vehicle-stably transfected SK-N-SH cells and siMDM2 or vehicle-stably transfected SH-EP1 cells that were co-transfected with either XIAP IRES RNA or control RNA. We seeded and cultured 200 cells with or without RNA transfection for 2 weeks, then stained the colonies and counted them under phase contrast microscopy. Data represent the mean of three independent experiments (bars ± SD, *p < 0.05, **p > 0.5). E, representative plate pictures for colony formation of SK-N-SH cells transfected with or without RNA expression plasmids as indicated.