Fig. 5

Reduced myogenesis and increased adipogenesis in SV40-trasformed, GFP-sorted Alk5 ^–/– cells. (a) Western blot analysis of major signaling pathway components in SV40-trasformed and GFP sorted Alk5 ^+/+ and Alk5 ^–/– cells treated with or without TGFβ1, n = 3 cell isolations from 6 pairs of lungs were used for each condition. (b–e) Representative images of Alk5 ^+/+ and Alk5 ^–/– cells treated with (c and e) or without (b and d) TGFβ1 for 48 hrs. Note the morphology difference between Alk5 ^+/+ and Alk5 ^–/– treated with TGFβ1 (c vs. e). (f) Quantitative PCR (Q-PCR) of mRNA isolated immediately after 48 hours of TGFβ treatment showed induced αSMA and Prrx1 mRNA by TGFβ1 only in Alk5 ^+/+ cells, while Tnc mRNA was induced in both Alk5 ^+/+ and Alk5 ^–/– cells. n = 2–3 cell isolations from 6 pairs of lungs per experiment, repeated once for each. (g and h) Oil Red O staining showed lipofibroblasts (LIFs) increased extensively in Alk5 ^–/– mesenchymal cells, cultured in 2 % low serum for 5 days. (i) Q-PCR showed increased LIF-related gene expression in Alk5 ^–/– cells cultured for 5 days. n = 4 cell isolations from 6 pairs of lungs per experiment, repeated 2 to 3 times. (j) Q-PCR showed increased Zfp423 and Wisp2 mRNA in Alk5 ^–/– cells. n = 5 to 6 cell isolations from 6 pairs of lungs per experiment, repeated 1 to 4 times. k. Gel image of Q-PCR products quantified in (l) of Zfp423 mRNA in Alk5 ^+/+ and Alk5 ^–/– cells, treated with or without TGFβ1 for 48 hrs. Zfp423 mRNA was decreased only in the Alk5 ^+/+ cells, treated with TGFβ1. n = 2 cell isolations from 6 pairs of lungs per experiment, repeated once for each. Error bars show standard error of the mean except for (l) which shows standard deviation. *P <0.01. Scale bars: e = 20 μm, h = 10 μm