Fig. 7: Pharmacological inhibition of eIF2α-ATF4 alleviates liver fibrosis.

a 8-week-old male C57BL/6 mice were administered with CCl₄ (0.5 ml/kg) or solvent control (corn oil) and simultaneously with or without ISRIB (5 mg/kg) twice weekly for 5 weeks. n = 5–7 mice per group. Western blots showing the expression of EMT and fibrotic marker genes in the liver tissues of mice with different treatments. The experiment was repeated three times. b qPCR analysis showing the expression of fibrotic genes in hepatocytes and HSCs isolated from the liver tissues from (a) (n = 3 biological replicates). c The representative images of Sirius red staining for liver tissues of (a) (n = 6 images derived from 3 mouse livers per group). d The hepatic collagen content of the liver tissues of (a) was determined by hydroxyproline quantification (n = 4 biological replicates). e 8-week-old male C57BL/6 mice underwent a sham (Sham) operation or surgery of common bile duct ligation (BDL), followed by biweekly administration of ISRIB (5 mg/kg) or solvent control for 4 weeks (n = 5 mice per group). Western blots showing the expression of EMT and fibrotic marker genes in the liver tissues of mice with different treatments. The experiment was repeated three times. f 8-week-old male C57BL/6 mice were administered with solvent control, TAA (150 mg/kg), or a combination of TAA and ISRIB (5 mg/kg) twice weekly for 5 weeks (n = 5 mice per group). Western blots showing the expression of EMT and fibrotic marker genes in the liver tissues of mice with different treatments. The experiment was repeated three times. g The hepatic collagen content of the liver tissues of e was determined by hydroxyproline quantification (n = 4 samples per group). h The hepatic collagen content of the liver tissues of (f) was determined by hydroxyproline quantification (n = 5 samples per group). i t-SNE representation of scRNA-seq analysis of HSCs from human liver of healthy donors (n = 5) and cirrhotic patients (n = 5). Cells from healthy or cirrhotic samples are highlighted in cyan or red, respectively. (adapted from GSE136103). j Violin plot showing the mRNA expression of ATF4 in HSCs of (i). k GSEA was performed with two genesets of ATF4 targets in a data set where genes were ordered by the fold-change of their expression comparing the HSCs of the cirrhotic patients and the healthy donors of (i). The geneset “ATF4 targets under TGFβ” (left) refers to genes regulated by ATF4 under TGFβ treatment in the LX-2 cells originating from the above bulk RNA-seq; The geneset “ATF4 targets under Tg” (right) refers to genes regulated by ATF4 under Tg treatment in the LX-2 cells also originated from the above bulk RNA-seq. The statistical significance (nominal P value) of the normalized enrichment score (NES) was generated by employing an empirical phenotype-based permutation test. l Spearman correlation of the Fib-4 scores and the expression of ATF4 in HSCs in the liver tissue from 15 cirrhotic patients. Determined by the intensity of IHC staining, the ATF4 expression in HSCs in these tissues was put into four categories: negative (1), low (2), medium (3), and high (4). Data are presented as mean ± s.e.m. Unpaired, two-tailed Student’s t tests were applied for (b, d, g, h, j). Kolmogorov–Smirnov tests were applied for (k). Spearman’s correlation was applied for (l). Source data are provided as a Source Data file.