Fig 2: LPS facilitates CIDEA expression by demethylating its promoter. (A) Data from the Meth Primer site predicted the CpG islands' location in the CIDEA promoter (Homo sapiens and Mus musculus). (B–C) Effect of LPS on CIDEA expression in a dose-dependent manner in hepatic cells (n = 5). (D–E) Different concentrations of LPS affected the total 5-mC levels in hepatic cells (n = 5). (F) Representative methylation-specific PCR (MSP) showed the methylation status of CIDEA CpG island after LPS (500 ng/mL) treatment in primary hepatocytes and HepG2 cells. Ratio (M/M+U) represents the methylated rate (n = 3). (G) LPS (500 ng/mL) affects the transcription activity of CIDEA (n = 3). (H–I) Quantitative ChIP reveals the enrichment of DNMT3B within the predicted region in LPS (500 ng/mL)-treated hepatocytes (n = 3). (J) Effect of SREBP-1c interference on CIDEA expression (n = 3). (K) The levels of SREBP-1c binding at the promoter of CIDEA in LPS (500 ng/mL)-treated hepatocytes (n = 3). (L) Effect of CIDEA knockdown with lentivirus expressing shRNA on LPS (500 ng/mL)-induced TG accumulation in primary hepatocytes and HepG2 cells pretreated with oleic acid (100 μM) (n = 3). Values are determined by the Student t-test when the 2 groups were compared. For 3 or more groups comparison, 1-way analysis of variance followed by Newman-Keuls multiple comparison test was used. Data are exhibited as mean ± standard error of the mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. 5-mC, 5-methylcytosine; siRNA, small interfering RNA.

Fig 3: DNMT3B is required for CIDEA expression. (A–B) Effect of DNMT3B overexpression (A) or DNMT3B knockdown (B) on CIDEA expression levels in primary hepatocytes and HepG2 cells (n = 3). (C–D) The CpG islands were detected through bisulfate sequencing (BSP) in the CIDEA promoter region (between −56 and −251bp, −146 and −254 bp from the transcription start site) after DNMT3B overexpression or knockdown. (E) The total 5-mC levels after DNMT3B overexpression (n = 5). (F–G) Quantitative ChIP analysis was used to measure the binding of DNMT3B (F) and 5-mC signals (G) at the promoter of CIDEA in DNMT3B forced expression hepatocytes (n = 5). (H) The transcription activity of CIDEA was verified after DNMT3B overexpression (n = 5). (I–J) Effect of CIDEA interference on TG content in LPS (500 ng/mL)-treated and DNMT3B knockdown hepatic cells that were pretreated with oleic acid (100 μM) for 24 hours (n = 5). Values are determined by the Student t-test when the 2 groups were compared. For 3 or more groups comparison, 1-way analysis of variance followed by Newman-Keuls multiple comparison test was used. Data are exhibited as mean ± standard error of the mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. 5-mC, 5-methylcytosine; siRNA, small interfering RNA.

Fig 4: DNMT3B overexpression decreases hepatic steatosis in LPS-infused and HFD-fed mice. C57BL/6 mice (male) were infected with AAV8-DNMT3B or a negative control via tail vein. Chow diet or HFD was then fed to mice for 8 weeks and infused with LPS (300 μg/kg/day) continuously during the first 4 weeks. (A) Expression efficiency of DNMT3B in mice liver. Body weight (B), liver weight (C), ALT and AST serum level (D) (n = 7). (E) Gross liver appearance (upper), Oil Red O (middle), H&E (middle), Sirius red staining (bottom), and (F) TG content measurement in the indicated group (n = 7). (G) Liver of inflammatory biomarkers (n = 5). (H) Effect of AAV8-DNMT3B on CIDEA expression level in the liver (n = 5). (I–J) Palmitate oxidation rate in hepatocytes isolated from mice liver with AAV8-DNMT3B infection (n = 5). Values are determined by the Student t-test when the 2 groups were compared. For 3 or more groups comparison, 1-way analysis of variance followed by Newman-Keuls multiple comparison test was used. Data are exhibited as mean ± standard error of the mean. ASM, Acid-soluble metabolites.