Fig 2: Hepatic Creg1 deletion aggravates inflammatory responses in ethanol-fed mice. (A-F) Creg1flox/flox and Creg1?hep mice were ethanol-fed for 10 days and administered a single binge of ethanol (Gao-binge model). The proinflammatory cytokines (A) TNFa, (B) IL-ß and (C) IL-6 were tested by using ELISA kits (n=4 per group). (D) F4/80 immunohistochemistry staining as a marker of macrophages and (E) MPO immunohistochemistry staining as a marker of neutrophils (n=4 per group). Scale bar = 50 µm. (F) Western blotting analysis to evaluate the activation of NF-?B signalling (n=3 per group). (G) mRNA levels of proinflammatory (Tnfa, Il6, Il1, F4/80, Ly6g, and Ccl2) genes (n=6 per group). ß-Actin served as the loading control. All data are represented as the mean ± SD. *p < 0.05, **p < 0.01 by two-tailed Student's t test. Tnfa, tumour necrosis factor alpha; Il6, interleukin 6; Il1ß, interleukin 1 beta.

Fig 3: Hepatic CREG1 expression levels are increased in ALD patients and ethanol-fed mice. (A) Liver tissues were obtained from patients with ALD and healthy controls. Immunohistochemistry analysis of hepatic CREG1 expression (n=3 per group). Scale bars: 50 µm. (B) Serum was obtained from intoxicated individuals and healthy controls. ELISA analysis of CREG1 expression in serum (n=43 per group). (C-E) Mice were pair-fed or ethanol-fed for 10 days and administered a single binge of ethanol (Gao-binge model). (C) Immunohistochemistry assessment of CREG1 expression (n=4 per group). (D) mRNA levels of hepatic CREG1 (n=4 per group). (E) Western blotting to measure hepatic CREG1 expression (n=3 per group). ß-Actin served as the loading control. All data are represented as the mean ± SD. *p < 0.05, **p < 0.01 by two-tailed Student's t test. CREG1, cellular repressor of E1A-stimulated genes 1.

Fig 4: CREG1 inhibits ethanol-induced activation of ASK1-JNK/p38 signalling. (A) Creg1flox/flox and Creg1?hep mice were ethanol-fed for 10 days and administered a single binge of ethanol (Gao-binge model). The protein levels of total and phosphorylated ASK1, TAK1, ERK, JNK, and p38 were assessed (n=3 per group). (B) AML-12 cells were transfected with or without CREG1 siRNA and subsequently treated with 100 mM ethanol and analysed by Western blotting for total and phosphorylated ASK1, JNK, and p38. (C) AML-12 cells were transfected with or without pEX3-CREG1 and subsequently treated with 100 mM ethanol and analysed by Western blotting for total and phosphorylated ASK1, JNK, and p38. ß-Actin served as the loading control. All data are represented as the mean ± SD. *p < 0.05, **p < 0.01 by two-tailed Student's t test. ASK1, apoptosis signal-regulating kinase 1, JNK, c-Jun N-terminal kinase, TAK1, TGF-ß-activated kinase 1, ERK, extracellular signal-regulated kinase.

Fig 5: Treatment with an ASK1 inhibitor abolishes alcoholic liver injury in Creg1?hep mice. (A-J) Creg1flox/flox and Creg1?hep mice were injected with the ASK1 inhibitor GS4997 or vehicle (DMSO) before ethanol feeding (Gao-binge model). (A) The serum levels of ALT in the indicated groups (n=5 per group). (B) Hepatic triglyceride (TG) and (C) total cholesterol (TC) levels (n=5 per group). (D) Representative HE staining and (E) Oil Red O staining of liver tissues from the indicated groups. Scale bar = 200 µm; scale bar = 50 µm. (F-I) mRNA levels of Fasn, Srebp1c, Il-1ß and Tnfa in the indicated groups (n=4 per group). (G) Protein levels of ASK1, JNK, p38, p65, I?Ba, C-Casp3 and phosphorylated ASK1, JNK, p38, and p65 in liver tissues from the indicated groups. ß-Actin served as the loading control. All data are represented as the mean ± SD. *p < 0.05, **p < 0.01 by two-tailed Student's t test.