FIGURE 9:
Treatment with pirfenidone and nintedanib impacts the RhoA signaling pathway in IPF cells. LL29 cells were treated with pirfenidone (1 mM) and nintedanib (1 μM) for 24 h. (A) The LL29 cells were then lysed, and Rnd3 levels analyzed by Western blot. (B) Quantification of Rnd3 expression from three independent assays. (C) The LL29 cells were then analyzed for p190 activity using a GST-RhoAQ63L pull-down assay. (D) Quantification of p190 activity from three independent assays. (E) LL29 lysates were then analyzed for the activation of RhoA measured through a GST-RBD pull-down assay as described under Materials and Methods. (F) Quantification of RhoA activity from three independent assays. (G) LL29 cells were lysed, and lysates evaluated by Western blot for FN, collagen I, SMA, and Erk2 levels. * p < 0.05 vs. (–) pirfenidone or (–) nintedanib as determined by a t test. (H) LL29 cells were plated onto FN-coated coverslips for 8 h and subsequently either untreated or treated with nintedanib (1 μM) or pirfenidone (1 mM) for an additional 16 h. F-actin staining was detected with Texas-red-labeled phalloidin.