Figure 4.

TGF-β₁ induces α–smooth muscle actin (α-SMA) mRNA and protein expression, which can be inhibited by SpA. IPF (A–D) or normal (C) fibroblasts were cultured without (solid bars) or with (open bars) TGF-β₁, and with the indicated concentrations of SpA (A and B) or 10 nM SpA (C) before harvesting for RNA or protein extraction at the appropriate time point. (A and C) α-SMA mRNA expression was analyzed by quantitative RT-PCR and calculated relative to the geometric mean of the housekeeping genes UBC/A2, using the ΔΔCT method. The data in A are expressed as fold stimulation relative to the untreated control sample, and represent means ± SD (n = 3 separate experiments), using one IPF fibroblast cell line. The data in C were calculated as percent inhibition of gene expression by SpA compared with that seen in the absence or presence of TGF-β₁ alone, and are shown as means ± SD from three IPF and three normal fibroblast lines (n = 3 per subject). (B) α-SMA protein expression at 72 hours was analyzed by Western blotting, with histone H3 as a loading control. (D) Immunofluorescent staining of α-SMA in fibroblasts, treated in the absence or presence of TGF-β₁ ± SpA (10 nM) for 48 hours, as indicated. Nuclei were counterstained with 7-amino actinomycin D (red). *P < 0.05, versus corresponding control sample. **P < 0.01, versus corresponding control sample.