Luciferase reporter assay

For luciferase assays, 293T cells were seeded to 24-well plates at a density of 3 × 10⁴/well. On the following day, cells were transiently transfected with the indicated luciferase reporter plasmids for 100 ng and pCDH/pCDH-HIF1α(∆ODD) for 500 ng, using T-Pro P-Fect Transfection Reagent (JT97-N005M). After overnight incubation, transfected cells were lysed with reporter lysis buffer (Promega) and assayed for firefly luciferase activity using a Multimode microplate reader, TECAN SPARK (TECAN). Briefly, the cells were harvested and washed with PBS, and then 100 µL of reporter lysis buffer (Promega, Cat# E3971) plus protease inhibitors (Roche) was added to the cells. The cells were scraped from the dish, and both the cells and solution were transferred to a microcentrifuge tube. Debris was pelleted via brief centrifugation, and the supernatant was transferred to a new tube. Then, 20 µL of cell lysate was mixed with 100 µL of Luciferase Assay Reagent, and the light produced was measured. The luciferase activity was normalized against the total protein concentration.