Transfection of HeLa cells for luciferase analysis

HeLa cells (100 000) per well (six-well plate) were transfected 18–24 h after plating with 0.2 µg of luciferase expression plasmids [luciferase expression is driven by either CMV (pGL3 pCMV, Promega) or L1 5′UTR promoters (52 (link))], 0.02 µg of the MT1 expression plasmid and 0.02 µl of the pBIND plasmid (Mammalian two-hybrid system, Promega), which expresses Renilla luciferase. pGL3-basic (Promega) was used as a negative control. DNA was transfected using 1 µl of Plus and 1.5 µl of lipofectamine reagents (Invitrogen). The transfection cocktail was replaced with regular media 3 h after transfection. Cells were harvested for luciferase activity analysis 48 h after transfection. Protein extraction was performed according to the manufacturer's protocol (Promega). Protein concentrations were determined using standard BSA approach. The same amount of protein for each sample was analyzed using Promega dual-luciferase detection system.