<i>SOX14</i> methylation status and expression analysis in the HeLa cell line.

<p><b>A</b>- Schematic illustration of <i>SOX14</i> promoter and positions of CpG islands 1, 2, 3 and 4 (presented in blue); positions of methylation specific PCR products 1 and 2 (MSP1 and MSP2) are shown in black lines. <b>B</b>- Methylation analysis of the <i>SOX14</i> promoter by methylation specific PCR (MSP) in HeLa cells. <b>C</b>- Methylation analysis of the <i>SOX14</i> promoter by MSP in NT2/D1 cells. M- primer pairs that anneal only to sequences that are methylated before bisulfite treatment; U—primer pairs that anneal only to sequences that are unmethylated. The product corresponding to MSP1 is 183 bp in length, while that corresponding to MSP2 is 118 bp in length. <b>D</b>- RT- PCR analysis of <i>SOX14</i> expression in HeLa and NT2D1 cells. <i>GAPDH</i> was used as a loading control. Three independent experiments were performed and one representative PCR is shown. NT2/D1 cells were used as a positive control for <i>SOX14</i> expression. m- DNA ladder, C- PCR negative control. <b>E</b>–Immunocytochemical analysis of SOX14 expression in HeLa and NT2/D1 cells. NT2/D1 cells were used as the positive control for SOX14 expression. Scale bar: 50 μm. <b>F</b>–Expression of <i>SOX14</i> following treatment with 5-azaC. HeLa cells were treated with 5μM 5-azaC for 72 h. Total RNA was subjected to RT-PCR in order to analyze <i>SOX14</i> mRNA expression. <i>GAPDH</i> was used for normalization. Three independent experiments were performed, and one representative PCR is shown. m- DNA ladder, C- PCR negative control.