2.6. Inhibition of DNA methylation and histone deacetylation in cell lines

Demethylation was induced in cancer cell lines with 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) (Millipore) as described before (Mossman et al., 2010; Zhang et al., 2007). Cells were incubated with 5‐aza‐dC at 10 μm concentration for 72 h, and the culture media were replaced every 24 h with fresh media containing 5‐aza‐dC. Control experiments were performed by the addition of DMSO solvent (Sigma‐Aldrich) and following the same procedure. Cell line treatments with histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (Selleck Chemicals, Houston, TX, USA) were carried out at 1 μm concentration for 24 h as previously published (Gill et al., 2013). Control experiments were treated by the addition of solvent alone. Following the completion of 5‐aza‐dC and TSA treatments, RNA from each sample was extracted for qRT‐PCR assays. Experiments were performed in four replicates.