Figure 4

Ketotifen treatment increased phosphorylation of YAP, decreased TAZ protein levels, and inhibited AKT phosphorylation. HDFa cells were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. (A, B) Gene expression of YAP (YAP1) and TAZ (WWTR1) were assessed using RT-qPCR and normalized to housekeeping gene HPRT. (C) Phosphorylated YAP at serine residue 127 and total YAP protein were measured using western blot and semi-quantitative assessments made by normalization to GAPDH. (D) TAZ protein was measured and plotted in the same manner. (E) Representative images of the blots are shown. (F) Phosphorylated AKT at serine residue 473 was measured in TGFβ1-treated fibroblasts, in the presence of ketotifen or AKT inhibitor, MK2206 (left). Representative blot images of phosphorylated AKT are on the right. Full-length blots are available in Supplementary Fig. 4. Data shown as mean ± SEM. n = 3 per treatment condition. *p < 0.05; **p < 0.01; ***p < 0.001. αSMA alpha-smooth muscle actin, AKT protein kinase B, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TAZ transcriptional coactivator with PDZ binding motif, TGFβ1 transforming growth factor-beta 1, YAP Yes-associated protein.