Firefly and renilla dual glo luciferase assay system

Human breast carcinoma cells (MDA-MB-231) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Mouse mammary carcinoma cells (4T1) were kindly provided by Dr. Jeong-Seok Nam (Gwangju Institute of Science and Technology, Gwangju, ROK). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (Cellgro, Manassas, VA, USA). TNFα was purchased from Sigma-Aldrich (St. Louis, MO, USA). The firefly and Renilla Dual-Glo^™ Luciferase Assay System was purchased from Promega (Madison, WI, USA). The pRL-null plasmid, which encodes Renilla luciferase, was also purchased from Promega.

MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (Corning Cellgro, Manassas, VA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Corning Cellgro). TNFα was purchased from Sigma-Aldrich (Saint Louis, MO, USA), and Pierce BCA Protein Assay Reagent was supplied by Thermo Scientific (Rockford, IL, USA). The Firefly and Renilla Dual-Glo^™ Luciferase Assay System was procured from Promega (Madison, WI, USA), and nitrocellulose membrane, from Bio-Rad Laboratories (Hercules, CA, USA).

Tissues were harvested from bladder specimens removed at the time of Transurethral resection of bladder tumor (TURBT) for primary and recurrent carcinoma of the bladder in the same patients. Cells were cultured at 37°C with 5% CO2 in RPMI 1640 supplemented with 10% v/v Fetal bovine serum (FBS) and penicillin (100 units/ml) /streptomycin (0.1 mg/ml). Recurrent bladder tumor cultures from three patients were sorted as cancer cells and stromal cells by CK7 antibody via FACS LSRII (BD Biosciences). Cancer cells were termed as PB1, PB2 and PB3 in primary cancers, and RB1, RB2 and RB3 in recurrent cancers. 293T cells were culture at 37°C with 5% CO2 in RPMI 1640 supplemented with 10% v/v FBS and penicillin (100units/ml)/streptomycin (0.1mg/ml). SB656933, SB202190, SB203580 and LY294002 were purchased from Calbiochem (San Diego, CA, USA) and Human recombinant GRO-α was from ProSpec Biology (East Brunswich, NJ, USA). The firefly and Renilla Dual-Glo™ Luciferase Assay System was purchased from Promega (Madison, WI, USA). The pRL-null plasmid, which encodes Renilla luciferase, was purchased from Promega.