Figure 2 | Cell Death & Disease

Figure 2

From: Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFβ-induced Nox4 expression and myofibroblast transdifferentiation

Figure 2

Nox4 inhibition with GKT137831 and Nox4 suppression with siRNA decrease dermal myofibroblast transformation. (a) hDFs were pretreated with vehicle or GKT137831 (20 μM) for 1 h before stimulation with TGFβ for 48 h. Immunofluoresence staining for αSMA (green) and filamentous actin stain with phalloidin (red) was performed. This experiment was repeated three times and the percentage of αSMA+ myofibroblasts were quantified over the three experiments. (b) hDF cells were pretreated with vehicle or GKT137831 (20 μM) for 1 h followed by TGFβ (10 ng/ml) for 24 h. Cellular mRNA was analyzed via quantitative real-time PCR (qRT-PCR) for changes in gene expression of SM22α, Nox4, fibronectin and Col1α1. (c) Collagen gel contraction assay was performed with hDF cells. Cells were treated with vehicle or GKT137831 (20 μM) in the presence or absence of TGFβ (10 ng/ml). Experimental groups were evaluated in triplicate. Change in gel surface area was determined after 48 h and is represented as the percentage of contraction of the gel. Experiments in panels (a−c) were repeated at least three times. Data are presented as mean±S.E.M. **P<0.05 versus −TGFβ; #P<0.05 versus vehicle+TGFβ treatment. (d) After electroporation of control or Nox4 siRNAs, hDF cells were stimulated with TGFβ (10 ng/ml) for 24 h. Total RNA was collected to analyze changes in gene expression by qRT-PCR. (e) hDF cells were electroporated with control or Nox4 siRNA and, after 3 days, were stimulated with TGFβ for 48 h. Immunofluoresence staining for αSMA (green) and phalloidin staining for filamentous actin (red) was performed to detect myofibroblasts. The percentage of cells that transdifferentiate to myofibroblasts was determined. The average of three experiments is presented in the bar graph. (f) qRT-PCR for changes in SM22α, fibronectin and Col1α1 mRNAs. (g) To detect changes in ROS, DCF-DA assay was performed on cells electroporated with control or Nox4 siRNA and incubated with or without TGFβ (10 ng/ml). As a positive control, hDF cells electroporated with control siRNA were treated with 4.8 nM H202. ROS detection assay was performed twice and each treatment group was evaluated in quadruplicates. Immunofluorescence staining in panel (d) and qRT-PCR experiments in panels (e and f) were performed three times each. All data are presented as mean±S.E.M. **P<0.05 versus −TGFβ; #P<0.05 versus control siRNA+TGFβ

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