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. 2021 Feb 22;6(4):e140180. doi: 10.1172/jci.insight.140180

Figure 2. Differential expression and activation of hepatic MLKL in mice exposed to Gao-binge ethanol diet compared with FFC diet.

Figure 2

For a model of NAFL/NASH Mlkl+/+ mice were allowed free access to a Western diet high in fat, fructose, and cholesterol (FFC) for 12 weeks or standard chow diets. For a model of ALD, mice were allowed free access to the Lieber-DeCarli ethanol diet or pair-fed control diet for 10 days followed by an acute gavage of ethanol or maltose, respectively. (A) Western blot analysis of MLKL was measured in livers from mice fed the FFC diet or Gao-binge ethanol model. A liver sample from an Mlkl–/– mouse fed the FFC diet was used as a negative control. n = 4–10 per group. (B) Paraffin-embedded liver samples were deparaffinized and stained for pMLKL. Nuclei were counterstained with hematoxylin. Arrowheads show phospho-MLKL (pMLKL)-positive areas. An Mlkl–/– mouse exposed to the high-fat diet feeding protocol was used as a negative control. Representative images are shown. n = 4–11 per group. Scale bar: 100 μm. P less than 0.05, assessed 2-tailed t test (A) and 2-way ANOVA (B); values with different alphabetical superscripts were significantly different from each other. (C and D) Oligomerization and subcellular localization of MLKL in the livers of mice were assessed in the cytosol, 10,000g pellets (P10) and isolated plasma membranes (PMs). Proteins were resolved by nonreducing PAGE and probed with antibody to MLKL. A longer exposure is shown in the right panel to better illustrate MLKL in the PM fraction. (C) Distribution of MLKL monomer and oligomer in liver of mice exposed to Gao-binge ethanol. (D) Distribution of MLKL monomer and oligomer in liver of mice exposed to Gao-binge ethanol compared with FFC diet. Representative images are shown. C, chow; E, Gao-binge; F, FFC; P, pair-fed.