Figure 3

(A) Cell count over time after treatment of PC-3 cells. VN/66-1 (10 μ M), MS-275 (5 μ M) and the combination VN/66-1+MS-275 (5+2.5 μ M) were used, respectively. P<0.001 for 18–72 h vs control (ANOVA) (two-way ANOVA was carried out and most time points were found to be significantly different for different treatments, but values are not reported here). (B) The percent of late apoptotic/necrotic cells as determined by Annexin V-FITC and PI positive staining by flow cytometry. ^***P<0.001 compared to control; ^###P<0.001 compared to VN/66-1; ^##P<0.01 compared to VN/66-1; ^{^^}P<0.01 compared to MS-275 (ANOVA). (C) RARβ mRNA expression after treatment of PC-3 cells for 24 h. ^*P<0.01 and ^**P<0.001 vs control and VN/66-1 alone (t-test). Data are mean (±s.e.) of three independent experiments (A–C). (D) Western blot analysis after treatment of PC-3 cells for 24 h (p21, acetyl H3, H4 and γ-H2AX) or 4 h (Bad, Bcl-2). Blots are representative of three independent experiments. Note: the same concentrations as stated for (A) were used for subsequent experiments.