Stable Transfection and Luciferase Assay

All of stable or transient transfections were performed using PolyJet™ DNA in vitro transfection reagent (SignaGen Laboratories, Rockville, MD) according to manufacturer’s instructions. Stable transfectants were selected with puromycin, hygromycin B or G418 for 3–4 weeks [26 (link), 27 (link)], and surviving cells from the antibiotics selection were pooled as stable mass transfectants, with at least two passages prior to utilization for further experiments. For the determination of p21 promoter-mediated luciferase activity, cells were transfected with the related luciferase reporter in combination with the pRL-TK vector (Promega, Madison, WI). After 24 h of transfection, luciferase activity was determined using a dual-luciferase reporter assay system kit (Promega, Madison, WI) as described previously [36 (link), 37 (link)]. The results were normalized by internal TK signal, and expressed as mean ± standard deviation from at least three independent experiments.