Prl vector expressing renilla luciferase

The reporter plasmid used contains the firefly luciferase gene under the control of the RNF126 promoter. A pRL vector expressing Renilla luciferase (Promega) served as an internal control. MDA-MB-231 cells (5×10⁴ per well) were seeded into 24-well plates and co-transfected with a reporter plasmid (100 ng) and the internal control vector (10 ng) in the presence or absence of U0126 (10 μm). Transfection was performed using Lipofectamine 2000 (Life Technologies). Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Luminescence was measured in a GloMax 20/20 luminometer (Promega).

The reporter assay was performed as previously described, but with minor modifications^{14 (link),21 (link)}. A pGL4.35 reporter vector containing the firefly luciferase gene under the control of a transcriptional regulatory unit comprising 9× Gal4-binding elements was purchased from Promega. A pRL vector expressing Renilla luciferase (Promega) served as an internal control. The HT1080 cells (1 × 10⁶) were seeded in a 90-mm dish and co-transfected with reporter plasmid (2.5 μg), internal control vector (250 ng), and pcDNA3 Gal4BD-HIF-1α CAD (727–826 amino acids) or pcDNA3 Gal4BD-HIF-2α CAD (769–870 amino acids) plasmid^{14 (link),21 (link)} (250 ng) using Lipofectamine 2000 (Thermo Fisher Scientific). Twenty-four hours after transfection, the cells (1 × 10⁴/well) were seeded in 96-well plates and treated with compounds at the indicated concentrations for 24 h. For the overexpression experiments, 50 ng/well of pcDNA3 V5-tagged Mint3, pcDNA3 V5-tagged FIH-1, or mock plasmid were transfected to HT1080 cells 2 h before compound treatment. Luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. Luminescence was measured using a GloMax 20/20 luminometer (Promega).