RASSF1A expression and promoter methylation in ACC cell lines. (A) Indirect immunofluorescence detection of RASSF1A protein (FITC – green) expression in SW-13, NCI-H295R and ACT-1 (a thyroid cancer cell line used as a positive control for RASSF1A expression) cells. Cell nuclei fluoresces blue due to DAPI fluorescence. (B) RASSF1A promoter methylation pattern in exponentially growing cultures of NCI-H295R and SW-13 cells as determined by Epitect methyl II PCR assay. Averages of percentage Hypermethylated (FHM) intermediate methylated (FIM), and unmethylated (FUM) CpGs are shown. (C &D) SW-13 cells were grown in the presence of varying (0, 0.1, 1, 5 and 10 μM) concentrations of 5-aza-2’-deoxycitidine for 48 hours and (C) Epitect methyl II assay was performed on genomic DNA to determine RASSF1A promoter methylation, and (D) RASSF1A mRNA expression was assayed by real-time qPCR. Average mRNA expression values of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP) were used for normalization.