Polyjet dna in vitro

Cell transfections were performed by using PolyJet DNA in vitro transfection reagent (SignaGen Laboratories) according to the manufacturer’s instructions. Surviving cells from the antibiotics selection were pooled as stable mass transfectants as described in our previous studies.3 (link), 51 (link), 55 (link) For the determination of p27 promoter-driven luciferase activity, FOXO1 promoter-driven luciferase activity or miR-196b promoter-driven luciferase activity, UMUC3(Nonsense), UMUC3(shATG7#1), and UMUC3(shATG7#2) cells were each transiently co-transfected with pRL-TK together with the related promoter-driven luciferase reporter. 24 hr after transfection, luciferase activity was determined using a luciferase assay system kit (Promega). For the determination of FOXO1 mRNA 3′ UTR activity, UMUC3(Nonsense), UMUC3(shATG7#1), and UMUC3(shATG7#2) cells were transiently transfected with pRL-TK together with FOXO1 mRNA 3′ UTR luciferase reporter or FOXO1 mRNA 3′ UTR mutant luciferase reporter. 24 hr after transfection, luciferase activity was determined using a luciferase assay system kit (Promega). The results were normalized by internal TK signal. All experiments were done in triplicate, and the results are expressed as mean ± SE.

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.