Fig. 3.

Demethylation agents 5′-aza-CdR and zebularine increase THRB expression through reduced THRB promoter methylation. A, The amounts of methylated (M) or unmethylated (U) THRB promoter determined by methylation-specific PCR analysis in human breast cancer cells (T47D, MCF-7, MDA-468) and thyroid cancer cells (SW1736, FTC-236, K-5, WRO, and FTC-133). Ba, Methylation of THRB promoter was decreased after treatment with the demethylating agents, 5′-aza-CdR (upper panel) and zebularine (lower panel) in the FTC-236 human thyroid cancer cell line; b, relative mRNA expression of THRB was increased by treatment with the demethylating agents in FTC-236 cells (data presented as mean ± sd and compared by Student's t test); c, protein abundance of TRβ was increased by treatment with demethylating agents in FTC-236 cells as shown by Western blot analysis of total cell lysates with or without treatment with demethylating agents as marked (upper panel). GAPDH was used as the loading control (lower panel). C, Relative to vehicle treatment, cell proliferation was inhibited by treatment with the demethylating agents in FTC-236 cells (data presented as mean ± sd). *, P < 0.01. D, Decreased Ki-67-positive cells after treatment with 5′-aza-CdR and zebularine in FTC-236 cells compared with vehicle treatment. Representative microphotographs of Ki-67 immunohistochemistry on paraffin-embedded cells after treatment with vehicle (a), 5′-aza-CdR (b), and zebularine (c). Cell proliferation index was determined by proportion of Ki-67-positive cells in FTC-236 cells (d). E, Relative to vehicle treatment, cell migration of FTC-236 was inhibited by treatment with demethylating agents, as determined by wound-healing assay (data presented as mean ± sd). *, P < 0.01.