Figure 5

5-aza-2′-deoxycytidine treatment induced demethylation and increased expression of EMILIN1 in fusion-positive rhabdomyosarcoma cells. (a) Cellular proliferation of six fusion-positive and five fusion-negative rhabdomyosarcoma cell lines in response to 5 -day treatment with varying concentrations of 5-aza-2′-deoxycytidine. At the indicated time points, triplicate determinations were quantified for each cell line using CellTiter 96 aqueous one solution cell proliferation assay according to the manufacturer’s instructions. (b) EMILIN1 promoter methylation at multiple CpG sites was measured by pyrosequencing in cells treated with 1 μM 5-aza-2′-deoxycytidine or vehicle alone for 5 days. Asterisks denote statistical difference at a P-value of <0.05. (c) EMILIN1 mRNA expression levels in fusion-positive and fusion-negative rhabdomyosarcoma cell lines. Quantitative reverse transcriptase-PCR was utilized to measure mRNA expression of test and control genes in triplicate. Upper panel, baseline EMILIN1 expression normalized to the level in RH5. Lower panel, changes in EMILIN1 mRNA expression levels in rhabdomyosarcoma cell lines treated with 5-aza-2′-deoxycytidine as compared with the corresponding control DMSO-treated cells. FN, fusion-negative rhabdomyosarcoma; FP, fusion-positive rhabdomyosarcoma; 5-aza-dC, 5-aza-2′-deoxycytidine.