p21WAF1 expression induced by U0126 is dependent on MyoD and/or myogenin. (A) Immunoblots of total lysates from RD cells transiently transfected with control-, MyoD- or myogenin-siRNA, or with a combination of MyoD- and myogenin-siRNA (MyoD+myogenin), and then left untreated (-) or treated with U0126 (+) for 2 days. (B) Immunoblots of total lysates from Puromycin-selected polyclonal population from RD cells transfected with the empty vector (CMV), MyoD- or myogenin-expressing vector. As a control, cells were left untransfected in the absence (C) or in the presence of TPA (TPA). Immunoblots were performed using specific antibodies capable of recognising MyoD, myogenin, p21WAF1 and α-tubulin. Densitometric analysis of bands provided quantification of MyoD and myogenin levels expressed as a fold increase over the control value (CMV) arbitrarily set at 1. (C) Luciferase assay of lysates from RD cells co-transfected with the empty vector (CMV), myogenin- or MyoD-expressing vector and the plasmid carrying p21WAF1 promoter (DM-Luc). Data show mean values ± s.e.m. of triplicates of a representative experiment. Similar results were obtained in two experiments.