Figure 7
HTS fibroblasts have increased propensity for myofibroblast transformation, which can be blocked with JQ1. (a) HTS and NBS fibroblasts were preincubated with vehicle or JQ1 (1 μM) for 1 h before being stimulated with TGFβ (10 ng/ml) for 48 h. Cells were then immunostained for αSMA (green) and co-stained for f-actin with phalloidin (red). αSMA+ myofiboblasts were quantified in three separate experiments. Data are presented as mean±S.E.M. *P=0.055 versus NBSF; **P<0.05 versus unstimulated cells; #P<0.05 versus TGFβ. (b) NBS and HTS fibroblasts were subjected to quantitative real-time PCR (qRT-PCR) analysis of myofibroblast gene expression changes after incubation with or without JQ1 (1 μM) and TGFβ (10 ng/ml). The experiment was repeated three times. Data are presented as mean±S.E.M. *P<0.05 versus NBSF; **P<0.05 versus unstimulated cells; #P<0.05 versus TGFβ. (c) BRD4 knockdown with siRNA was performed in HTS fibroblasts before TGFβ (10 ng/ml) stimulation for 24 h. Cellular mRNA was extracted and qRT-PCR was performed for BRD4-dependent myofibroblast genes. This experiment was repeated three times. Data are presented as mean±S.E.M. **P<0.05 versus control siRNA−TGFβ; #P<0.05 versus control siRNA+TGFβ. (d) XChIP analysis for Smad3 and BRD4 on the Nox4 promoter was performed on NBS and HTS fibroblasts incubated with or without TGFβ (10 ng/ml) for 6 h. Fold change is calculated relative to NBSF−TGFβ sample. XChIP experiment was performed twice and similar results were observed. Data are presented as mean±S.E.M. (e) XChIP for CDK9 binding to the Nox4 promoter. Experiment is carried out as in panel (d)
