Figure 4

Reactivation of TFPI-2 using epigenetic drugs. (A) RT-PCR analysis of TFPI-2 expression in OCSL cells treated with 5-azaDC, TSA, or both. 0.5A, 0.5 μM 5-azaDC; 5A, 5 μM 5-azaDC; TSA, 0.25 μM TSA; 0.5A + T, 0.5 μM 5-azaDC and 0.25 μM TSA; 5A + T, 5 μM 5-azaDC and 0.25 μM TSA. The mRNA level of TFPI-2 in OCSL was compared with that of the control cell line IOSE, which was assigned a value of 1. Error bars indicate standard deviations. (B) Methylation levels of OCSL and OC2 cells treated with 5-azaDC, TSA, or both. The DNA of OCSL and OC2 was bisulfite converted and the percentage of TFPI-2 methylation was determined using the pyrosequencing assay. The TFPI-2 methylation level is expressed as a percentage of that in IVD (given as 100%). The DMSO- and drug-treated groups were compared using Student’s t-test; P < 0.05 (*) was considered statistically significant. (C) Histone modifications at the promoter region of TFPI-2 in OCSL cells. ChIP assays were performed with antibodies against H3K9-Ac and H3K9-me3 in OCSL cells. Regions of the TFPI-2 promoter for quantitative ChIP-PCR assay are indicated as R1 and R2. The binding of H3K9-Ac and H3K9-me3 antibodies to regions R1 and R2 was measured by the quantification of ChIP DNA against a standard curve generated from input DNA. The binding level of each antibody in OCSL treated with 5 μM 5-azaDC and 0.25 μM TSA was compared with that of the cell treated with DMSO, which was assigned a value of 1.