The Cignal Lenti NFAT Reporter Assay was used to determine PKA/Ca2+ pathway activity. Cignal Lenti NFAT Reporter Assay (luc) (4 x 105 TU) and Cignal Lenti Renilla Control (luc) (1 x 105 TU) cotransduced around 10,000 normal human pulmonary artery smooth muscle cells (PASMC). After 48 hours transduction, medium was changed to assay medium. After 54 hours transduction, cells were treated with 10 ng/ml PMA and 0.5 µM ionomycin for 18 hours. Dual-luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were performed in triplicate and the standard deviation is shown.
The Cignal Lenti RBP-Jk Reporter Assay was used to measure Notch signaling activity in C6 (rat glioma) cells. Cignal Lenti RBP-Jk Reporter Assay (luc) (2 x 105 TU) and Cignal Lenti Renilla Control (luc; 2 x 104 TU) transduced around 20,000 C6 cells. After 24 hours transduction, medium was changed to complete medium. After 48 hours transduction, cells were infected with 100 MOI of recombinant adenoviruses expressing constitutive active Notch1 (Ad-NICD) or 100 MOI of recombinant adenovirus expressing GFP (Ad-GFP). Dual-luciferase assay was performed 18 hours after infection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were performed in triplicate and the standard deviation is shown.
The Cignal Lenti NFκB Reporter Assay was used to measure NFκB pathway activity in thymocytic cells (D1; Murine T-cell leukemia cells). Cignal Lenti NFκB Reporter Assay (luc) (2.5 x 105 TU) transduced around 10,000 D1 cells. After 48 hours transduction, cells were treated with 20 ng/ml recombinant mouse TNFα (hTNFα) protein for 18 hours. Luciferase assay was performed and promoter activity values are expressed as arbitrary units. Experiments were performed in triplicate and the standard deviation is shown.
Cignal Lenti HIF-1 Reporter (luc) (1.5 x 105 TU) transduced around 15,000 HepG2 cells (24 hours before transduction 7,500 cells were plated per well of 96-well plate) with and without Cignal Lenti Renilla control (3 x 104 TU). After 42 hours of transduction, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 48 hours of transduction, cells were treated with 300 µM of CoCl2 for 18 hours. Luciferase or dual-luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. The utilization of the dual-luciferase system (cotransduction of Cignal Lenti Renilla Control along with Cignal Lenti Reporter Assay (luc)) improves the fold induction change in hypoxia signaling activity, as normalization of firefly luciferase activity with Renilla luciferase activity corrects for cell death caused by the treatment.
The Cignal Lenti NFkB Reporter Assay (luc) was used to generate an NFkB pathway sensor cell line for the study of the NFkB signal transduction pathway. The NFkB sensor cell line was developed by transduction of HEK-293 cells with the Cignal Lenti NFkB Reporter Assay (luc), followed by selection of a clonal population that maintained stable chromosomal integration of the lentiviral vector provirus and responded strongly to stimuli known to activate the NFkB pathway. The generation of stable HEK-293 NFkB sensor cell line was confirmed by testing the cell line with 10 ng/ml of recombinant hTNFα after 1, 5, 10, and 15 passages of the cell line. Stimulation of the NFkB pathway by hTNFα results in up to a 100-fold increase in expression of the reporter gene even after 2 months of cell culture.
