Fig 1: PPARα target gene expressions were repressed by RXRα protein reduction in let7b/c2ΔHep and let-7 sponge AAV-transduced mice(A) Heatmap of PPARα target genes identified by differential gene expression analysis of RNA-seq data from let7b/c2+/+ and let7b/c2ΔHep livers after HFD feeding.(B and C) mRNA analysis by qRT-PCR of PPARα target genes involved in fatty acid oxidation and cell proliferation (B) and lipid accumulation and glucose metabolism (C) in HFD-fed let7b/c2+/+ and let7b/c2ΔHep livers.(D) Western blot analysis for PPARα target genes in HFD-fed let7b/c2+/+ and let7b/c2ΔHep liver lysates.(E) Western blot analysis of PPARα and RXRα protein expression in nuclear fractions isolated from let7b/c2+/+ and let7b/c2ΔHep hepatocytes.(F–H) Western blot analysis of RXRα and the densiometric quantification in whole-liver lysates from let7b/c2ΔHep- mice (F), let-7 sponge expressing AAVinflected mice (G), and pre-let-7c-1--AAVinfected mice (H).Data are presented as mean ± SEM (n = 4–5 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001
Fig 2: RNF8 is decayed by let-7 miRNA, and RXRα protein is ubiquitinated by RNF8 E3 ubiquitin ligase(A) Predicted let-7 miRNA binding sites in Rnf8 mRNA.(B–F) Western blot analysis and densitometric quantification of RNF8 protein (B, D, and F) and qRT-PCR of Rnf8 mRNA (C and E) in let7b/c2+/+ and let7b/c2ΔHep (B and C); EGFP and let-7 sponge AAV-transduced (D and E); EGFP and pre-let-7c-1 AAV-transduced (F) livers treated with HFD feeding.(G) 3′ UTR reporter assays in HepG2 cells transfected with Rnf8 wild-type or mutant 3′ UTR reporter constructs and a let-7c mimic expression vector.(H and I) Western blot analysis (H) and densitometric quantification (I) of RXRα expression in Rxra- and Rnf8-transfected Hepa-1 cells.(J and K) Fold change of Rnf8 (J) and Rxra (K) mRNA by qRT-PCR analysis in Rxra- and Rnf8-transfected Hepa-1 cells.(L) Western blot analysis and the densitometric quantification of RXRα in Rxra- and Rnf8-transfected Hepa-1 cells treated with the proteasome inhibitor MG-132.(M) Ubiquitination assays for Rxra- and Rnf8-transfected and MG-132-treated Hepa-1 cells. RXRα was immunoprecipitated and polyubiquitin detected by anti-ubiquitin antibody. RXRα expression was confirmed in whole-cell lysate as input.(N) Scheme of 3-step inhibition for PPARα/RXRα pathway that the current study demonstrates.
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