Luciferase Assay for hTERT Regulation

Luciferase assays were performed as previously described by Gazon et al. [27 (link)]. Briefly, 293T cell line was used to set up the protocol, and then, HuT78 and MyLa cells were transfected with a plasmid DNA mixture containing 100 ng·µL⁻¹ of pGL3‐hTERT‐378‐Luc reporter plasmid [28 (link)], 100 ng·µL⁻¹ of pActin‐βgal, and the indicated amount of pAD/WT1‐IRES‐nAMcyan (gift from Edward McCabe, Addgene [Watertown, MA, USA] #29756). HuT78 and MyLa were electroporated using Gene Pulser XCell Electroporation Systems (Bio‐Rad). Forty‐eight hours post‐transfection, cells were washed with cold PBS and then lysed in 1× passive lysis buffer (Promega). Luciferase and β‐galactosidase assays were both performed in a Spark 10M Multiplate Reader (Tecan, Männedorf, Switzerland) with Genofax A Kit and Genofax B Kit (YELEN, Ballaison, France), and Galacto‐Star Kit (Life Technologies, Grand Island, NY, USA), respectively, as described by the manufacturer. Each experiment was performed in triplicate, and Luciferase activities were normalized for transfection efficiency based on β‐galactosidase. After WT1 overexpression, the levels of hTERT mRNA were also evaluated by qRT‐PCR.