Figure 1.

TGFβ induction of IRE1α is critical for HSC activation. A, LX-2 cells were stably infected with an shRNA targeting procollagen 1α1 (shCollagen 1α1) or an NT control. Cells were treated with 5 ng/ml TGFβ for 0, 1, 2, 4, or 24 h, followed by assessment of phosphorylation and protein levels of IRE1α, fibronectin (FN), αSMA, and collagen I. HSC70 served as a loading control. Quantification is shown adjacent. B, LX-2 cells were transfected with an siRNA targeting SMAD2 (siSMAD2) or a nontargeting siRNA (siControl). 24 h post-transfection, cells were serum-starved and treated with TGFβ for 0, 1, 2, 4, or 24 h. Cell lysates were harvested, and levels of phosphorylated and total IRE1α, SMAD2/3, and HSC70 (loading control) were assessed by immunoblotting. Quantification is shown adjacent. C, primary HSCs were isolated from IRE1α^fl/fl mice and infected with adenovirus expressing Cre recombinase (AdCre) or LacZ as a control. 48 h postinfection, cells were treated with TGFβ or vehicle. Cell lysates were harvested and analyzed by immunoblotting for FN, collagen I, αSMA, and HSC70 (loading control). Quantification is shown adjacent. D and E, LX-2 cells were pretreated with the IRE1α inhibitor 4μ8C (15 μm) for 1 h, followed by TGFβ or vehicle for 24 h. Cell lysates or mRNA were harvested and analyzed by either immunoblotting (D) for FN, collagen I, and HSC70 (loading control) or qPCR (E) to assess fibronectin, procollagen 1α1 and 1α2, αSMA, or PPARγ expression. Quantification for D is shown below the blots. Statistics were performed using ANOVA followed by Tukey post hoc analysis (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars, S.E.