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. 2018 Feb 2;68(2):347–358. doi: 10.1136/gutjnl-2017-315348

Figure 4.

Figure 4

Liver injury and inflammation are tempered by hepatocyte-specific TonEBP deficiency. (A–D) TonEBPfl/fl; Albumin cre+/− mice (HKO) and their TonEBPfl/fl; Albumin cre−/− (WT) littermates were injected with DEN or PBS. Livers and serum samples were analysed 48 hours later. (A) Livers from the WT animals were immunoblotted for TonEBP and Hsc70. (B) Serum ALT levels in PBS-treated (n=5) and DEN-treated animals (n=8). Mean+SEM. *P<0.05 compared with WT. (C) TUNEL signal in hepatic tissues. (D) RT-qPCR analyses of TonEBP, inflammatory genes (COX-2, TNFα, IL-1β, IL-6) and proliferation markers (Ki-67, cyclin D1) in livers of DEN-treated animals. mRNA abundance was expressed relative to WT group. P, PBS; D, DEN. Mean+SEM, #P<0.05 compared with corresponding P. *P<0.05 compared with WT. (E) Immunoblotting of liver samples, three from each genotype, for TonEBP, PCNA, Hsc70, caspase-3, C-Casp-3, COX-2 and iNOS. (F,G) HKO and WT animals were injected with PBS or LPS (5 µg/kg) in combination with D-galactosamine (400 mg/kg) (LPS). The animals were analysed 4 hour later. (F) Serum levels of ALT and AST. (G) RT-qPCR analysis mRNA levels. mRNA abundance was expressed relative to WT, PBS group. Mean+SEM, *P<0.05 compared with WT. #P<0.05 compared with PBS. (H–J) The animals were fed with Con or EtOH for 10 days. (H) Serum ALT, AST and LDH levels. *P<0.05 compared with WT. #P<0.05 compared with corresponding Con-fed animals. (I) RT-qPCR analyses of TonEBP and inflammatory genes in livers. (J) Representative images of H&E of hepatic tissues from animals from (H). ALT, alanine transaminase; AST, aspartate aminotransferase; C-Casp-3, cleaved caspase-3; iNOS, inducible nitric oxide synthase; Con, control diet; COX-2, cyclo-oxygenase-2; DEN, diethylnitrosamine; EtOH, ethanol diet; HKO, hepatocyte-specific TonEBP knockout; IL, interleukin; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; mRNA, messenger RNA; PBS, phosphate-buffered saline; TNFα, tumour necrosis factor alpha; TonEBP, tonicity-responsive enhancer-binding protein; WT, wild type.