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. 2015 May 8;6(24):20177–20189. doi: 10.18632/oncotarget.3896

Figure 5. ZEB1 elevates the methylation level of 14-3-3σ promoter.

Figure 5

A. 14-3-3σ mRNA levels represented as fold change were detected with real-time RT-PCR by normalizing to GAPDH as endogenous control and the expression level from Tca8113 was set as 1. 14-3-3σ protein levels were detected by western blotting. B. The location of the primers for MSP analysis of 14-3-3σ gene. MSP analysis determined the 14-3-3σ promoter methylation levels in TC cell lines. U: unmethylated primers; M: methylated primers. C. 14-3-3σ mRNA levels were detected by real-time RT-PCR in TC cells treated with 5 μM 5-aza-2′-dC and the expression level from control-treated cells was set as 1. 14-3-3σ protein levels from related treated cells were detected by western blotting. vs control, *p < 0.01. D. A schematic representation of ZEB1 binding site in the 2kb putative 14-3-3σ promoter and the first base of the 2kb strand is defined as ‘1’. ZEB1 mRNA and protein levels were detected by real-time RT-PCR and western blotting respectively. ChIP-qPCR results for the ZEB1 binding to the 14-3-3σ promoter in TC cell lines. vs the other cell lines, *p < 0.01. E. ChIP-qPCR results for the ZEB1 binding to the 14-3-3σ promoter in ZEB1 expression modulated cell lines by transfection of plasmids or siRNA. ZEB1 and 14-3-3σ protein levels were examined by western blotting. F. MSP results showed the change of methylation level in the 14-3-3σ promoter in ZEB1, β-catenin and GSK3β expression modulated cells with transfection of plasmids or siRNAs. G. ZEB1 mRNA levels were determined by real-time RT-PCR in β-catenin and GSK3β expression modulated cells by transfection of plasmids or siRNAs. *p < 0.01.