Fig. 5: Liver and gWAT inflammation are downregulated by dietary caloric input and tumor growth.

A qPCR analysis of liver Kupffer cells/macrophage population: F4/80 expression. Immunoregulation in liver lysates was evaluated by Western blot in control (tumor-free) and tumor-bearing samples from CR, Chow, or HFHS-fed mice. B Representative immunoblotting of Toll-like receptor 4 (TLR4), phosphorylated IkappaB kinase (IKK) (S176/180), IKKα, and inducible nitric oxide synthase (iNOS) in liver samples. eEF2 was used as a loading control. (C–E) Western blotting densitometries quantifications of C TLR4/eEF2, D pIKK (S176/180)/IKKα, and E iNOS/eEF2. The results are from three independent experiments (n = 3). F, G qPCR analysis of gWAT macrophage infiltration, F F4/80, and G Tnfa expression. Immunoregulation in gWAT lysates was evaluated by Western blot in control (tumor-free) and tumor-bearing samples from CR, Chow, or HFHS-fed mice. H Representative immunoblotting of TLR4, iNOS, and TNFα protein expression in gWAT lysates. β-actin was used as a loading control. I–K Western blotting densitometries quantifications of I TLR4/Actin, J iNOS/β-actin, and K TNFα/β-actin. The results represent three independent experiments (n = 3). Plotted values are mean ± S.E.M. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001. For all panels, a two-way ANOVA was performed, followed by Tukey’s post-test. qPCR reactions for each sample were conducted in duplicate and all tissue samples were analyzed.