CD133 expression is repressed by DNA methylation in prostate cancer cell lines. (A) qRT-PCR analysis (relative to HPRT, calibrator = P4E6) of CD133 expression in prostate cell lines treated with 1 μM 5-Aza-2'-deoxycytidine for 96 hours. (N.D. = expression undetectable after 40 cycles, n = 2; ± SD, * = p < 0.05; ** = p < 0.01 in a paired t-test, p-value not calculated in cell lines with non-detectable levels of CD133). (B, C) FACs analysis of CD133 expression in prostate cell lines treated with 1 μM 5-Aza-2'-deoxycytidine for 96 hours. Live cells were stained with CD133/2(293C)-APC antibody (Miltenyi Biotec) (CD133-APC) or without any antibody (No Antibody) and analysed by FACs. For each dot plot, X axes: FITC channel fluorescence (not stained); Y axes: APC channel fluorescence (CD133 or No Antibody control); Percent of CD133+ cells is indicated and the results are summarised in C. (D) Dot-plot showing expression (qRT-PCR, relative to HPRT, calibrator = Bob) and methylation of CD133 in a panel of prostate cell lines grouped on the basis of their origin (benign or cancer) and their culture conditions (low FCS = 0%-2%; high FCS = 5%-10%).