Figure 5. NREP is downregulated by palmitate-induced steatosis in HepG2, and NREP regulates hepatic lipid metabolism.

(A) NREP protein levels in HepG2 cells treated with BSA or palmitate for 24 hours (n = 3 independent experiments). (B) Quantification of NREP protein levels. (C) NREP knockdown (KD) in HepG2 cells at protein levels (n = 3). (D) Representative oil red staining in HepG2 cells with NREP KD challenged with palmitate for 24 hours (n = 3 independent experiments; original magnification, ×400; scale bar: 50 μm). (E and F) Triglyceride (E) and cholesterol (F) content quantification in HepG2 cell lysates after stimulation for 24 hours with 500 μM palmitate (n = 3 independent experiments). (G and H) RNA sequencing selected enriched pathway analyses of upregulated (G) and downregulated genes (H) in HepG2 cells with NREP KD compared with scramble (n = 3 per group). (I) Heatmap representation of differently expressed genes involved in cholesterol biosynthesis, AKT signaling, apoptosis, fibrosis, and cell cycle. (J) Basal signaling analyses in lysates from HepG2 cells treated with scramble (left) or NREP KD (right) (n = 3 independent experiments). (K) Protein levels of indicated proteins in HepG2-scramble and NREP KD treated with BSA or palmitate for 24 hours in the presence of AKT inhibitor (MK-2206) or DMSO (n = 2 independent experiments). Significance was determined by 2-tailed unpaired t test in B, and 1-way ANOVA with Holm-Šidák multiple-comparisons test in E and F. **P < 0.01; ***P < 0.001. Data are expressed as means ± SEM.