Figure 2.

RIP3 kinase activity was required for FFC-induced phosphorylation and oligomerization of MLKL in mouse liver and liver injury. Rip3^+/+ and Rip3^K51A/K51A mice were allowed free access to chow or FFC diet for 12 weeks. (A) Immunohistochemistry staining for pMLKL in paraffin-embedded liver sections. Images were acquired using a 10X objective. Representative images are shown from n=5-6 per group. (B) Subcellular fractions (cytosol, P10 (10,000 g pellet) and plasma membrane (PM) were isolated from liver, proteins separated by SDS-PAGE under non-reducing condition, and immunoblotted (IB) with anti-MLKL antibody. Enrichment of sub-cellular fractions was verified by probing for Na⁺/K⁺-ATPase (plasma and intracellular membrane marker) and β-actin (cytosolic marker). Blots are representative from n = 3 for PM and 6 for cytosol and P10 independent experiments. Western blots were semi-quantified on Image J. Values for MLKL oligomer in the P10 fraction were 0.05 ± 0.02^a for chow-fed and 0.32 ± 0.14^b for FFC-fed in Rip3^+/+ and 0.05 ± 0.03^a for chow-fed and 0.12 ± 0.04^a for FFC-fed in Rip3^K51A/K51A (p<0.05). Values for MLKL oligomer in the PM fraction were 0.07 ± 0.03^a for chow-fed and 0.24 ± 0.06^b for FFC-fed in Rip3^+/+ and 0.05 ± 0.02^a for chow-fed and 0.07 ± 0.02^a for FFC-fed in Rip3^K51A/K51A (p<0.05). (C) Hematoxylin and eosin (H&E) staining of liver sections. Images were acquired at 10X magnification. (D) ALT/AST concentration in plasma and hepatic triglyceride content in liver homogenates. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different from each other, n = 5-6 per group. p <0.05, assessed by ANOVA.