Figure 5

Inhibition of CDK9 with Can508 or CDK9 knockdown with siRNA decreases myofibroblast transformation. (a) hDFs were pretreated with Can508 (30 μM) for 6 h before stimulation with TGFβ (10 ng/ml) for 48 h. Immunofluorescence for αSMA (green) and phalloidin staining for f-actin (red) was performed. αSMA+ myofibroblast cells were quantified in three separate experiments. (b) Myofibroblast gene expression changes were analyzed via quantitative real-time PCR (qRT-PCR) in hDF cells pretreated with Can508 and stimulated with TGFβ (10 ng/ml) for 24 h. (c) Collagen contraction assay was performed on hDF cells treated with vehicle or Can508 (30 μM) in the presence or absence of TGFβ (10 ng/ml). Change in surface area is reported as the percentage of contraction of the gels. Experiments in panels (a–c) were repeated at least three times. Data are represented as mean±S.E.M. *P<0.05 versus vehicle−TGFβ; **P<0.05 versus −TGFβ; ^#P<0.05 versus vehicle+TGFβ. (d) hDF cells were electroporated with control or CDK9 siRNA before being stimulated with TGFβ (10 ng/ml) for 48 h. Top panel, whole-cell extracts extracts were assayed for CDK9 expression by western blotting. GAPDH was used as a loading control. Western blotting was repeated twice with similar results being observed. Bottom panel, qRT-PCR analysis of CDK9 mRNA. Data are presented as mean±S.E.M. of at least three experiments. (e) Cells were immunostained for αSMA and also stained for f-actin with phalloidin (red) to determine the myofibroblast population. Average of five experiments is represented in the bar graph. (f) mRNA was assayed for gene expression by qRT-PCR. Data are presented as mean±S.E.M. of at least three experiments. *P<0.05 versus control siRNA−TGFβ; **P<0.05 versus −TGFβ; ^#P<0.05 versus control siRNA+TGFβ