Fig. 6

Role of Pdgfrα. (a) Quantitative PCR (Q-PCR) analysis showed induction (fold increase over controls) of Pdgfrα and Pdgfrβ mRNAs in response to 48 hours of TGFβ1 treatment only in Alk5 ^+/+ cells. Controls were treated exactly the same with the exception of bovine serum albumin in place of TGFβ. For each condition, the RNA was isolated from combined triplicate wells. For Pdgfrα, n = 2 cell isolations from 6 pairs of lungs per experiment, repeated 2 to 3 times. For Pdgfrβ, n = 3 cell isolations from 6 pairs of lungs per experiment. All conditions were initiated and analyzed after the same incubation period. (b) Progressive increase in Pdgfrα and Pdgfrβ mRNAs, without TGFβ1-induction during spontaneous differentiation of Alk5 cell lines in culture for 0, 2, and 5 days. For each condition, the RNAs were isolated from combined triplicate wells. n = 2 cell isolations from 6 pairs of lungs per experiment, repeated once for each. In both (a) and (b), RNA was isolated at the indicated time point and Q-PCR was performed immediately after. Note higher levels in Alk5 ^–/– compared to Alk5 ^+/+ cells on both day 2 and day 5. (c) Western blot analysis confirmed higher PDGFRα protein in three independent samples of Alk5 ^–/– cells on both day 2 and day 5, compared to Alk5 ^+/+. n = 3 independent experiments. β-ACTIN was used as a control. (d–f) Alk5 ^+/+ cells were treated with or without Imatinib at 5 μm and 10 μm for 5 days, mRNA for PDGF signaling target genes (d), myofibroblast-related genes (e) and lipofibroblast-related genes (f) were analyzed by Q-PCR (n = 2 cell isolations from 6 pairs of lungs per experiment for (d) and n = 3 cell isolations from 6 pairs of lungs per experiment for (e) and (f). Error bars show standard deviation in (d) and standard error of the means in (e) and (f). *P <0.05