Fig. 3: ATF4 positively regulates EMT program in HSCs and is required for HSC activation.

a Western blots showing the expression of mesenchymal marker genes in LX-2-shCtrl and LX-2-shATF4 cells treated with or without TGFβ for 2 days. The experiment was repeated four times. b Western blots showing the expression of mesenchymal marker genes in LX-2 cells treated with or without ISRIB (1 mM) and TGFβ (0, 1, and 5 ng/ml). The experiment was repeated three times. c Western blots showing the expression of mesenchymal marker genes in LX-2 cells overexpressing ATF4 treated with or without increasing concentrations of TGFβ (0, 0.5, 1, and 5 ng/ml) for 2 days. The experiment was repeated three times. d qPCR analysis of mesenchymal marker genes mRNA expression in LX-2 cells overexpressing ATF4 treated with or without increasing concentrations of TGFβ (0, 0.5, 1, and 5 ng/ml) for 2 days (n = 4 biological replicates). e Western blot showing the expression of fibrotic genes in LX-2-shCtrl and LX-2-shATF4 cells treated with increasing concentrations of TGFβ (0, 5, and 10 ng/ml) for 2 days. The experiment was repeated four times. f qPCR analysis of mRNA expression of fibrotic genes in LX-2-shCtrl and LX-2-shATF4 cells treated with TGFβ for 2 days (n = 3 biological replicates). g qPCR analysis of mRNA expression of fibrotic genes in LX-2 cells overexpressing ATF4 for 4 days and then treated with increasing concentrations of TGFβ (0, 0.5, and 1 ng/ml) for 2 days (n = 4 biological replicates). h qPCR analysis of mRNA expression of fibrotic genes in LX-2 cells treated with solvent control (Ctrl), TGFβ (5 ng/ml) for 2 days (TGFβ), or pretreated with ISRIB (1 mM) for 3 days followed by treatment with TGFβ (5 ng/ml) and ISRIB (1 mM) for another 2 days (TGFβ + ISRIB) (n = 3 biological replicates). Data are presented as mean ± s.e.m and representative of at least three independent experiments. Unpaired, two-tailed Student’s t tests were applied for (d) and (f–h). Source data are provided as a Source Data file.