Figure 3.

The actin cytoskeleton architecture in TGF-β-treated HBFs was modulated by GW501516 alone or in combination with GSK0660. HBFs derived from asthmatic patients (n = 3) were cultured in a serum-free medium containing GW501516 and GSK0660 alone or in combination, and in the absence or presence of TGF-β₁ (5 ng/mL) for 24 h. (A) Representative images of HBFs immunostained for vinculin (green) and F-actin (red) with visualization of DNA (blue) are presented; scale bar = 50 µm. Vinculin-rich focal adhesion sites are shown enlarged in boxes; scale bar = 25 µm. (B) F-actin fluorimetry was quantified in relation to DNA fluorescence and is presented in the graph (n = 3, 260 cells/condition). (C) The lengths of vinculin-rich focal adhesion sites were measured and are presented in the graph. (D,E) The contents of focal-adhesion-related proteins vinculin and talin, were determined via in-cell ELISA (n = 8; each condition in triplicate). (F) The relative expression of TLN (talin) in HBFs (n = 3) cultured for 24 h under the conditions described above was determined using real-time PCR. The results are presented as 2^−ΔΔCt mean values in relation to the control gene (GAPDH). RCU–relative colorimetric units. The results are presented as the mean ± SEM. Statistical significance was tested using the nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001.