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. Author manuscript; available in PMC: 2011 Feb 2.
Published in final edited form as: Mol Cancer Ther. 2010 Feb 2;9(2):336. doi: 10.1158/1535-7163.MCT-09-0589

Figure 3. Both promoter CpG dinucleotides methylation and mRNA differentially splicing contribute to the decreased Keap1 expression in DU-145 cells.

Figure 3

(A) Schematic representation of sodium bisulfite sequencing of 5-mCpG at the Keap1 promoter in DU-145 cells, human peripheral WBCs, and PrEC cells. Each circle represents an individual CpG site. Closed circles, methylated CpG sites; open circles, unmethylated sites. The beginning and end of each amplicon relative to the TSS of the longest Keap1 isoform is indicated. The region from -393 to -93 in the PrEC genomic DNA contained many non-informative CpG dinucleotides that showed deviation from the consensus virtually bisulfite-converted sequence despite having high rates of non-CpG conversion. (B) Left panel, real-time RT-PCR analysis of Keap1, NQO1, and Gclm expression. Right panel, Amplification of full-length Keap1 transcript. PCR products were resolved on agarose gel. (C) Amplification of full-length keap1 transcript in prostate cancer cells (Left panel) and Schematic representation of Keap1 WT transcript and DU-145 derived alternatively spliced products (right panel). E2 to E6 represent relative position of exon-2 to exon-6, open boxes stand for 5′ and 3′ untranslated regions. (D) Aberrantly spliced Keap1 transcripts code for non-functional truncated Keap1 proteins. The WT and differentially spliced Keap1 transcripts cloned in pDNA3.1 expression vector were transfected into DU-145 cells, and Keap1 protein expression was measured by immunoblot assay (Left panel). Truncated Keap1 proteins are unable to suppress Nrf2 depdendent NQO1 reporter activity. (right panel).