Figure 7. WNK1 mediates TGFβ-induced myofibroblast differentiation in a kinase-independent manner.

A, Representative images of western blot analyses of the effects of siRNA targeting WNK1 (siWNK1) and the WNK kinase inhibitor WNK463 on myofibroblast markers and intracellular signaling pathways in HLF. HLF were transfected with siRNA targeting ANO1 (siANO1) or control RNA (siCont) for 24 hrs, serum starved for 48 hrs, pretreated with or without WNK1 inhibitor WNK463 (10 μM) for 1 hr, followed by treatment with TGF-β (1 ng/ml) or vehicle control for 48 hrs. Cell lysates were then probed for the immunoreactivity of WNK1, collagen 1A1 (Col1A1), fibronectin (FN), smooth muscle α-actin (SMA), p-MLC, MLC, p-AKT, and AKT. B, Quantification of the immunoreactivity of WNK, Col1A1, FN, SMA, p-MLC, and p-AKT. Data are mean IR values ±SD normalized to β-actin loading control and TGFβ group within the same experiment. *p<0.05, **p<0.01, ***p<0.001, vs. TGF-β group, Kruskal-Wallis test with Dunn’s post hoc correction. Brace brackets, #p<0.05; ##, p<0.01, are the results of preplanned comparisons of the TGFβ effect in cells treated with siWNK1 or the WNK1 inhibitor WNK463, t-test.