Luciferase assay

The luciferase assay method to measure the direct DNA methylation effect on gene expression has been described in detail elsewhere [12, 13]. In short, 1500-bp fragments of human promoters from LAMP2 and RPS4X were cloned into a CpG-free luciferase vector (pCpGL-basic) [27] and amplified by GenScript (GenScript USA Inc., Piscataway, NJ, USA). The constructs where then either mock methylated or methylated in vitro using the methyltransferases SssI, HhaI or HpaII (2.5 U/μg DNA) (New England Biolabs), which methylate cytosines in the following context: CG, GCGC and CCGG, respectively. Mouse myoblasts (C2C12) cultured in DMEM (4.5 g/L glucose) supplied with 10% FBS and 1% PS in a 96-well plate were co-transfected with either 150 ng DNA/well (LAMP2) or 50 ng DNA/well (RPS4X) of the pCpGL vector constructs together with a Renilla luciferase control reporter vector (Promega, Madison, WI, USA) using FuGeneHD (Promega). Luciferase and Renilla activity were measured 48 h post transfection in cell lysate using Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was calculated as the ratio between the reporter gene firefly luciferase and the control vector Renilla luciferase.