Pgl4 firefly luciferase reporter

To study the role of the Oct-1Z isoform in the development of cellular response to stress, the pGL4-Firefly Luciferase Reporter Vectors with Cellular Stress Response Elements– pGL4.42[luc2P/HRE/Hygro] and pGL4.39[luc2P/ATF6 RE/Hygro] (Promega) were used. IMR32-Empty Vector and IMR32-Oct-1Z cell lines were transfected with the above-mentioned plasmids together with the pRL-Tk plasmid (Promega) using the TransFast reagent according to the manufacturer’s instructions. After transfection, cells were cultured in the complete MEM medium at 37 °C with 5% CO₂ for 24 h. After the end of the culturing period, cells were subjected to hypoxia or ER stress for 24 h as described above. Then, Dual Luciferase assay was carried out using the Dual-Glo Luciferase Kit (Promega). Luciferase signal was detected using the GloMax96 device with the 0.5 s integration time.

HEK293T cells were plated in 12-well plates at the density of 50,000 cells ml⁻¹. When confluent, cells were transfected using Fugene-HD (Promega) with a combination of (1) pGL4 Firefly luciferase reporter (Promega, E6651), (2) pRL Renilla luciferase control reporter (Promega, E2241), and (3) FLAG-hSP7 or FLAG-hSP7^R316C or GFP control (VectorBuilder, EF1A as a promoter, vector IDs are VB190819-1114pvu, VB190819-1115xzu, and VB180924-1105fst) plasmids. Ostn_En2 sequence was synthesized with gBlocks Gene Fragments (IDT) (See sequence information in Supplementary Data 10). SacI and BglII restriction enzymes (NEB R0156S, R0144S) were used to cut both the pGL4 plasmid and the Ostn_En2 fragment. In all, 48 h later, cells were disassociated by adding Passive Lysis Buffer (Promega, E1910) and were gently rocking on an orbital shaker for 15 min. In all, 20 µl cell lysate was transferred to 96-well black polystyrene microplates (Corning). In all, 50 µl of Luciferase Assay Reagent II was dispensed to each well with the reagent injector. The sample plate was placed in the luminometer to measure the Firefly luciferase signal. In all, 50 µl of Stop & Glo Reagent was then dispensed to each well. The plate was placed back to the luminometer to measure Renilla luciferase activity. Luciferase experiments were performed in biologic triplicate, and all experiments were repeated at least twice.