This effect was dose dependent RSV 0. 1 uM had a minimal effect, com parable to untreated till cells, while the highest concentra tion, RSV 25 uM, showed an important action on proliferation control. In Figure 2B, viability assay graph showed the absence of cell mortality in all treatment conditions. A very important Inhibitors,Modulators,Libraries support to those data were the mor phological changes observed in cells treated with 25 uM of RSV the cells seem to lose their characteristic circular shape, typical of the active proliferation phase, to achieve a new elongated morphology. Phase contrast images, collected at day 3 of growth curve, confirmed those morphological features morphological changes in cell size and shape are compared in detail, emphasizing the analogy between DM cells and 25 uM RSV treated cells.

Most Cyclins Inhibitors,Modulators,Libraries expression seems to decrease with the onset of differentiation, when cells are blocked in G1 phase. To achieve additional confirmation of data ob tained from the growth curve, viability test and morpho logical studies, we performed quantitative Real Time PCR during proliferation phase, to prove an actual decrease in Cyclins expression levels. As shown in the panel, RSV treatments cause a significantly down regulation in Cyclins expression, following DM control condition, in respect to GM time 0 control. To verify the absence of RSV cytotoxic effects on C2C12, we evaluated in Western Blot analysis the pro tein levels of the apoptotic marker p53 during pro liferation phase, showing how RSV treatment does not modify p53 protein amount in re spect to GM control condition.

Phase contrast images in Figure 3C, collected at 24 h and 72 h of proliferative phase, illustrated the morphological changes in RSV treated cells with respect to control. Furthermore, to corroborate Inhibitors,Modulators,Libraries RSV action on cell cycle regulation, we measured the protein content of cell cycle regulator p21 during proliferative phase. RSV treatment seems to cause a significant de crease in p21 protein levels with respect to control. The lower protein content in RSV treated cells with respect to growth control is comparable to differentiation control cells. Since p21 promotes cell cycle exit and induces cellular differentiation, we might suppose that RSV could induce cell cycle arrest and differentiation. To investigate RSV action on differentiation induction, we determinated protein amount of two early MRFs MyoD and Myf 5, key markers of differentiation induc tion.

Figure 4A elucidated Inhibitors,Modulators,Libraries the significant increase of Myf 5 and MyoD protein levels after RSV stimulation. In addition, we studied morphological changes in myo blasts through MyoD and Myf 5 Immunofluorescence analysis during proliferative phase. Knowing that MyoD and Myf 5 represent important markers for Inhibitors,Modulators,Libraries early CC5013 myogenesis stage and regulates skeletal muscle commitment, these results prove that RSV can advance differentiation induction.