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. 2017 Jan 19;8(9):15071–15084. doi: 10.18632/oncotarget.14748

Figure 3. XAF1 methylation in glioma cell lines.

Figure 3

(A) Schematic promoter region of XAF1 with the location of MS-HRM primers used for this analysis and sequencing primers used by Byun et al. [46] (B) XAF1 promoter methylation in 16 GB cell lines analyzed by MS-HRM. The cell lines were ordered by the percentage methylation of the analyzed XAF1 promoter fragment starting with the lowest methylation value (from left to right). MS-HRM was carried out in technical duplicates of bisulfate-converted DNA of each cell line. The dotted line indicates the threshold of methylation which was applied for grouping. Samples with methylation values above 33% were considered as “methylated”. (C) XAF1 mRNA expression in 16 GB cell lines as determined by quantitative RT-PCR. The expression (relative to GBP61) of XAF1 mRNA is shown, normalized to ACTB and ENOX2. Expression in each cell line was detected in technical triplicates (D) The grouping of the 16 GB cell lines according to XAF1 methylation status. Cell lines with a methylation value above 33% were considered to be methylated (M), whereas those with a methylation level of ≤ 33% were set as unmethylated (UM). Statistical significance for the difference in both groups was tested by a two-tailed t-test (p-value < 0.0001, (****)). (E) XAF1 protein expression in selected GB cell lines with HSP90 loading control. The XAF1 promoter methylation percentages as determined by MS-HRM are indicated below the blot.