Figure 4

Demethylation activates COX-2 promoter activity in HBV-positive cells. (A) Cells were seeded at a density of 1 × 10⁶ cells/10 cm dish and transfected with pCOX-2-Luc and pRL-TK. 5-aza-CdR was added to a final concentration of 1, 2, or 4 μM at 6 hours post-transfection. Cells were allowed to grow for another 24 hours for qRT-PCR, and 48 hours for luciferase assays and Western blots. Luciferase data were normalized to that of the Renilla luciferase value, and are presented as mean ± SD, n = 3. (B) Genomic sequencing data of the COX-2 promoter in HepG2 cells. Potential methylation sites are indicated with vertical lines. (C) Genomic DNA from HepG2 and HepG2.2.15 cells was treated with sodium bisulfite and PCR amplified for the COX-2 promoter. Amplicons were cloned and sequenced, and each row of circles represents a single sequenced clone. The solid circles indicate methylated cytosines, while the open circles indicate unmethylated cytosines. (D) Nuclear extracts were prepared from HepG2.2.15 cells and were mixed with a ³²P-labeled unmethylated NF-AT probe, or with an in vitro methylated NF-AT probe, and analyzed by EMSA. Excess non-labeled NF-AT probe was used as a competitor. wt: wild-type; methyl: methylated.