Promoter hypermethylation and loss of CD133 gene expression in colorectal cancers

Figure 1 Schemes of the promoter region of CD133. A: Schemes of the CD133 gene 5′ terminus; B: Distribution of CpG dinucleotides in a fragment of the CD133 gene harboring full P2 (250 bp) promoter and 5′-UTR exon 1B (The citation from Tabu et al[5] and Pleshkan et al[11]).

Figure 2 Expression analysis of CD133 gene in 32 colorectal cancer cell lines. A: Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the CD133 gene in 32 colorectal cell lines; B: Quantitative differences of CD133 expression by real-time PCR analysis.

Figure 3 Methylation analysis of CD133 gene in 32 colorectal cancer cell lines by methylation specific-PCR (MS-PCR). Lanes M and U denote that the product amplified by primer recognizing a methylated sequence and the product amplified by primer recognizing an unmethylated sequence, respectively.

Figure 4 Analysis of methylation status of promoter CpG islands of CD133 by clonal sequencing. A: Representative sequence diagrams of DNA sequencing analysis in (a) HT-29 (unmethylated) and (b) SW480 (methylated) cell lines. M: Methylated site; U: Unmethylated site; B: DNA sequencing analysis of 32 CpG dinucleotides. The numbers at the top show the CpG sites of amplified DNA by bisulfite sequencing primers. Circle represents CpG dinucleotides; closed circle: Methylation; open circle: Unmethylation.

Figure 5 Expression analysis after treatment with 5-aza-2’-deoxycytidine (5-Aza). A: RT-PCR analysis of CD133 expression in 11 cell lines with or without 5-aza-2’-deoxycytidine treatment; B: Representative results of real-time PCR analysis revealing CD133 expression in 5 cell lines with or without 5-aza-2’-deoxycytidine treatment.