Prl tk renilla reporter vector

A5, a murine cutaneous spindle cell line derived from a DMBA/TPA-treated SPRET/Out-R × CBA F1 mouse carcinoma, and C5N, a non-tumorigenic murine keratinocyte cell line isolated from a Balb/C mouse, were obtained from Allan Balmain (Zoumpourlis et al. 2003 (link)). These lines were chosen for study because they are of the relevant murine tissues to study candidates for Skts5 (keratinocyte or SCC derived). A5 was grown in Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Transient transfections were performed using Lipofectamine LTX and Plus reagents (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. Cells were plated in triplicate and transfected at 60-80% confluency. Mock transfection and pGL3 promoter empty vector transfections were included as controls. For luciferase assays, cells were co-transfected with a Promega pRL-TK renilla reporter vector. To determine transfection efficiency, we performed co-transfection of constructs with a Clontech pEGFP NI vector and assessed for GFP-positive cells. Transfection efficiency was around 60% for all cell lines and plasmids evaluated.

Cells were transfected at 70% confluence using Lipofectamine2000® Reagent (ThermoFischer Scientific), according to the manufacturer’s guidelines. In all transfections the pRL-TK Renilla reporter vector (Promega) was used as an internal control. Cells were lysed and Renilla and firefly luciferase activities were measured using the Dual-Luciferase® Reporter Assay System (Promega) and a Tecan Infinite M1000 microplate reader. Luciferase activities were normalized to Renilla internal control luminescence.