Figure 4.

9-ING-41 blocks TGF-β mediated fibroblast to myofibroblast transition. Fibroblasts (normal, A,B and IPF, C,D) were treated with various doses of 9-ING-41 (10-0.5 µM) in serum free media. Cells were then treated with TGF-β for 48 h. Conditioned medias and cell lysates were then resolved by SDS-PAGE and immunoblotted for collagen (Col-1), α-SMA, GSK-3β, and tyrosine-216 phosphorylated GSK-3β. Under control conditions, TGF-β induced Col-1 and α-SMA protein in NF and IPF fibroblasts (A and C). 9-ING-41 (10 and 5 µM) significantly blocked TGF-β mediated induction of α-SMA and Col-1 in NF and IPF cells. Tyr-216 phosphorylation of GSK-3β (pGSKt) was likewise reduced by pretreatment with 9-ING-41 in both NF and IPF fibroblasts. Graphed data are the means of n = 3 independent experiments. Images are representative of 3–4 independent experiments. Total RNA was isolated from TGF-β treated cells in the presence or absence varying doses 9-ING-41 (10–0.5 µM). Changes in α-SMA and collagen 1 expression were then determined by qPCR analyses (B and D). GAPDH was used as the reference gene. Data are expressed as mean ± SEM. n = 3 independent experiments. *Denotes p < 0.05 compared to TGF-β control. Normal (E) and IPF (F) fibroblasts were treated with varying doses of TDZD-8 (40-5 µM) prior to the addition of TGF-β. Cell lysates and conditioned media were then resolved via SDS-PAGE and probed for changes α-SMA and collagen. β-actin was the loading control. TDZD-8 modestly reduced α-SMA and collagen induction by TGF-β at the highest dose (40 µM). Images are representative of two independent experiments.