Figure 4

Effect of combinations of cytostatics with decytabine on the cell cycle and apoptosis regulators. (A), Analysis of CCNE1, ATM, p53 and CASP3 mRNA expression by semi-quantitative RT-PCR method after 72 h incubation of Colo-205 cells with cytostatics, decytabine and their combinations at concentrations as indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase – internal control. (B), Immunoblotting analysis of the level of pro-apoptotic and cell cycle regulatory proteins following 72 h incubation of colorectal cells with cytostatics (5-FU, oxaliplatin), decytabine and their combinations at indicated concentrations. A representative blot of 2–3 ones for each particular protein is shown. The β-actin was used as a gel loading control. (C), Comparison of the relative amount of pro-apoptotic and cell cycle regulatory proteins, in samples shown in panel B, estimated by blot scanning and quantification of band intensities with the QuantiScan ver. 3.0 (Biosoft, UK) densitometry analysis software. The protein levels were normalized according to the gel loading control (β-actin) and presented as a percentage of control (level of particular protein in untreated cells). For particular protein, two to three blots were analyzed (n = 2 to 3). Each bar represents the mean ± SD; asterisks indicates significance at p < 0.05, for comparison with control.