Figure 3. In vitro mammary fibroblast activation by TGF-β1 is directly inhibited by ibuprofen.

Treatment conditions are: 0.5 TGF-β1 = 0.5 ng/ml TGF-β1, 5 TGF-β1 = 5 ng/ml TGF-β1, Low IBU =10 μg/ml ibuprofen, High IBU = 30 μg/ml ibuprofen (HIBU). (A) Mammary fibroblast morphology when cultured on plastic (left panel) or within floating collagen pads (right panel). Dashed lines show the outline of the cells. (B) α-Smooth muscle actin (αSMA) gene expression in primary mammary fibroblasts cultured on plastic, within floating collagen pads, or from freshly sorted mammary PDGFRα⁺ fibroblasts, n = 6–10 per condition. (C) Morphologic evidence of TGF-β1–induced fibroblast activation in the floating collagen pad culture model. (D) Increased Col1a1 and Cox2 gene expression in fibroblasts treated with TGF-β1, n = 4–7 per condition. (E) The ability of TGF-β1–treated fibroblasts to contract collagen pads (top) is suppressed by ibuprofen (bottom) and (F) data quantification, n = 5 per condition. (G) Fibroblast morphology with TGF-β1 treatment in the presence or absence of ibuprofen. (H) Col1a1 and Cox2 gene expression in fibroblasts with TGF-β1 treatment in the presence or absence of ibuprofen, n = 5 per condition. Scale bars: 100 μm (A, C, and G) and 1 cm (E). All gene expression data are normalized to Actb and then normalized to the control groups in each experiment. TGF-β1 and ibuprofen combination treatment studies were repeated 5 times. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.0001 by 1-way ANOVA with Tukey correction. For data normalized to control, statistics were performed using matched 1-way ANOVA with Tukey correction on the raw data that are not normalized to control. Data represent mean ± SEM. NS, not significant.