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Figure 3 | Molecular Cancer

Figure 3

From: Promoter methylation of IGFBP-3 and p53 expression in ovarian endometrioid carcinoma

Figure 3

Aberrant promoter methylation and transcriptional regulation of p53 at IGFBP-3. (a) MSP shows IGFBP-3 promoter methylation in OEC cell lines. Upper panel: schematic diagram showing CpG islands in the IGFBP-3 promoter and the location of primers used (M, methylated; U, unmethylated). Arabic numbers indicate the location of nucleotides relative to the transcription start site (TSS) (+1). Vertical lines represent the position of the CpG dinucleotides. Lower panel: MSP in P0 and P4 sublines. U indicates unmethylated (158 bp) and M indicates methylated (129 bp) PCR products. The Sss I treated A549 (lanes 1 and 2) and H1299 (lanes 4 and 5) were used as methylated controls, A549 as unmethylated control (lanes 6 and 7), and Genomic DNA without bisulfite conversion as negative control (lanes 12 and 13). (b) Real-time PCR analysis of IGFBP-3 mRNA showing restoration of IGFBP-3 expression in P4. P0 and P4 cell lines were treated with 0, 0.1, 0.5 or 2 μM of 5-aza-dC for 8 days. Culture media was changed to serum free media for 24 hours before IGFBP-3 analysis. Lower panel shows Western blotting corresponding to IGFBP-3 expressions in P0 and P4 culture media after 5-aza-dC treatment. Similar amount of culture media from equal number of cells were loaded in each lane. (c) Luciferase activity after transfection with wild type IGFBP-3 promoter (-253 ~+61)-pGL3 luciferase reporter construct (pGL3-wt IGFBP-3). Luciferase activity was normalized against renilla activity. Transfection with pGL3-basic vector (pGL3) was used as negative control. Transfections were carried out in triplicate and were done in three independent experiments.

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