Fig 1: MLT regulated methylation of CES1 promoter though SIRT1-mediated DNMT1 deacetylation. (A) WB was used to assay DNMT (DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L expression after treatment with various concentrations of MLT. Densitometry and statistical analysis. Representative images are shown. (B) Luciferase reporter assays were performed after co-transfection of 22RV1 and 293T cells with a pGMLR-CMV luciferase reporter and CES1 promoter (CES1-WT) vector or a blank vector (CES1-MUT) plasmid with a DNMT1 overexpression or blank vector plasmid. (C) CES1 protein levels were determined by Western blotting in cells treated with DNMT1 inhibitor 5-aza-dC (0 µM, 5 µM, 10 µM, and 20 µM) for 4 days. Densitometry and statistical analysis. Representative images are shown. (D) DNMT activity assay kits were used to assay the DNMT activity in 22RV1 and C4-2 cells treated with various concentrations of MLT. (E) ChIP-PCR assays were performed to detect direct binding of DNMT1 to the CES1 promoter regions in 22RV1 and C4-2 cells. (F) WB was used to detect SIRT and DNMT1 protein expression levels in the C4-2 and 22RV1 cells treated with MLT (1 mM) 48 h after transfection with siMT1. Densitometry and statistical analysis. Representative images are shown. (G) WB was used to detect the expression of Ces1, Sirt1, and Dnmt1 in the animal CRPC models with a circadian rhythm disorder. Densitometry and statistical analysis. Representative images are shown. (H) After transfection with siSIRT1 or siNC, 22RV1 and C4-2 cells were treated with various concentrations of MLT. The cell lysate was immunoprecipitated with anti-DNMT1 antibody, and the precipitated proteins were assayed by Western blotting with anti-SIRT1 and anti-acetyl-lysine (anti-AcK) antibodies. Representative images are shown. The data from a representative of three independent experiments are shown. *p < 0.05, **p < 0.01, and ***p < 0.001. Abbreviation: ns, no significance