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. Author manuscript; available in PMC: 2007 Nov 17.
Published in final edited form as: Biochem Biophys Res Commun. 2006 Sep 22;350(2):385–391. doi: 10.1016/j.bbrc.2006.09.069

Fig.4.

Fig.4

GW7845 inhibits TGFβ1-dependent induction of ECM genes and activation of Smad3 without inducing toxicity in human HSC. LX-2 cells were cultured in MDI for 72h, pretreated overnight with vehicle or different doses of GW7845 or SB 431542 (10μM) and then stimulated with TGFβ1 (100 pM). Cultures were harvested after 1h (for Smad3 analysis) or 12h (to evaluate PAI-1, collagen). Cell lysates were immunoblotted with antibodies against PAI-1 and P-Smad3, membranes were reprobed for β-actin, Smad2/3 to confirm equal protein loading (A, D). Total RNA was extracted, subjected to reverse transcription, and then assessed for expression of PAI-1 and α (1) | collagen mRNA by real time PCR. In each sample, mRNA levels of PAI-1 or collagen were normalized to GAPDH (B, C). The results are expressed as relative fold induction compared to control. Data shown were as mean ± SEM of three independent experiments, each of which analyzed samples from triplicate wells. *p<0.05 versus control. (D) LX-2 cells were cotransfected with a PAI-1–luciferase reporter and PRL-CMV-renila control plasmid and treated with SB 431542 (10μM), GW7845 (1μM), or DMSO ± TGFβ1 for 24h. Luciferase activity was normalized to PRL-CMV activity, and the activity of mock-transfected cells was subtracted from all values. The results shown are the means ± SEM of triplicate wells. * p<0.05 versus control, # p<0.05 versus group treated with TGFβ1. (E). To assess PPARγ agonist cytotoxicity, LX-2 cells growing in MDI were re-plated onto 96-well plates (0.5×104 cells/well) for 24h. The cells were serum-starved for 24h in DMEM while treated with various doses of GW7845, or 0.2% Tween-20. LDH activity in the medium was measured. After subtracting LDH values in the negative (untreated) control cultures from the other values, a cell injury rate was calculated ({LDH value in treated cultures minus LDH value in untreated cultures} divided by 24h). Results are expressed as percentage of the cell injury rate in the positive control (PC) cultures that were totally lysed following Tween-20 treatment (F).