renilla luciferase internal control vector prl-tk  (Promega)

Journal: PLoS ONE

Article Title: Downregulation of miR-200a Induces EMT Phenotypes and CSC-like Signatures through Targeting the β-catenin Pathway in Hepatic Oval Cells

doi: 10.1371/journal.pone.0079409

Figure Lengend Snippet: (A) Predicted alignment of miR-200a and a potential binding site at the 3′-UTR of rat CTNNB1 mRNA (624–630 nt) by TargetScan. (B) Effect of miR-200a on CTNNB1 expression determined by a luciferase reporter assay. WB cells were co-transfected with anti-miR-200a (or anti-miR-control) and the pGL3-CTNNB1-wt (or pGL3-CTNNB1-mut) vector. Data are normalized by the ratio of Firefly and Renilla luciferase activities measured at 48 h post-transfection and are shown as the mean ± SD; n = 3; **, p<0.01. (C) Expression of β-catenin, c-myc and cyclin D1 in WB-miR-NC and WB-anti-miR-200a cells detected by western blot analysis. (D) Cellular localization of β-catenin visualized by immunofluorescence staining in WB-miR-NC and WB-anti-miR-200a cells. Nuclear counterstaining was performed by DAPI (magnification×200). (E) The activity of β-catenin signaling determined by a luciferase reporter assay using the wild-type (TopFlash) or mutant (FopFlash) TCF4-reporter plasmids. Data are presented as fold increases in firefly luciferase over Renilla activity from three independent experiments; *, p<0.05.

Article Snippet: Briefly, one day before transfection, cells were plated in a 24-well plate and were then transiently transfected with 100 ng of β-catenin-responsive firefly luciferase reporter plasmid TopFlash (Millipore) or negative control FopFlash (Millipore), as well as 10 ng of internal control pRL-TK Renilla luciferase vector (Promega) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Activity Assay, Mutagenesis