Figure 4

JQ1 treatment and BRD4 suppression with siRNA block myofibroblast transdifferentiation. (a) hDF cells were preincubated with 1 μM JQ1 or vehicle for 1 h before stimulation with TGFβ (10 ng/ml) for 48 h. Immunofluoresence staining for αSMA (green) and phalloidin staining for filamentous actin (red) was used to detect myofibroblasts. Myofibroblast cells (αSMA+) were quantified in three separate experiments. (b) hDF cells were preincubated with 0.5 or 1 μM JQ1 for 1 h and then stimulated with TGFβ (10 ng/ml) for 24 h. mRNA was analyzed via quantitative real-time PCR (qRT-PCR) to determine myofibroblast gene expression changes. (c) Collagen gel contraction assay was performed with hDF cells treated with vehicle or 1 μM JQ1 in the presence or absence of TGFβ (10 ng/ml). Gel surface area was measured at time 0 and 48 h and change in surface area is reported as the percentage of contraction of gel. The assay was performed in triplicate. Experiments in panels (a−c) were repeated at least three times. Data are reported as mean±S.E.M. *P<0.05 versus 0 μM JQ1−TGFβ; **P<0.05 versus −TGFβ ^#P<0.05 versus vehicle+TGFβ. (d) hDF cells were electroporated with control or BRD4 siRNA. After 72 h, cells were incubated with or without TGFβ (10 ng/ml) for another 48 h. Knockdown efficiency of BRD4 was determined via western blotting and qRT-PCR. β-Actin was used as a loading control. Western blotting analysis was repeated twice. (e) αSMA immunostaining (green) and phalloidin staining for filamentous actin (red) was performed to quantify myofibroblasts (aSMA+ cells). Myofibroblast cells were quantified in four separate experiments. (f) qRT-PCR analysis for myofibroblast genes. Data are presented as mean±S.E.M. *P<0.05 versus control siRNA−TGFβ; **P<0.05 versus −TGFβ; ^#P<0.05 versus control siRNA+TGFβ