Figure 6

Expression levels of different cell cycle regulators following E2F1 and cyclin E1/E2 depletion and effect of the combined administration of different siRNAs on BrdU incorporation. (A) Three d after transfection, the protein levels of different cell cycle regulators were measured in protein extracts obtained from CSMCs of donor 2: shown are representative blots. ^#All the numbers reported below the blots derive from band quantification normalized to GAPDH and are expressed as a ratio of siGL2 treatment; for ppRb (hyperphosphorylated pRB) and cyclin A2, whose phosphorylation status and protein levels, respectively, were modified, cumulative data are reported as ratio to GAPDH; data are expressed as means ± SEM, n = 3, ⁺P < 0.05 compared with control. (B) mRNA levels of cyclin A2 and Cdk2 were measured in CSMCs treated with siE2F1-1324 in total RNA extracts obtained from both donors (28S transcript levels served as normalizator); results are expressed as means ± SEM, n = 6; *P < 0.05 compared with control. (C) CSMCs were treated by a mix of the different siRNAs (115 or 76 nmol/L each in the case of a combination of two or three siRNA, respectively) and CSMC proliferation evaluated by BrdU incorporation; the effects of the combined administration were compared with an equimolar concentration of each of the single siRNAs (230 nmol/L each) 3 d after transfection; NTC, nontreated cells; siGL2, control siRNA treated cells; cumulative data from donor 1 and 2 CSMCs are expressed as means ± SEM, n = 3.