Cell Viability and Luciferase Activity Assessment in Human Renal Epithelial Cells (293T) Following Drug Treatment

Human renal epithelial cell line 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and seeded in 96-well plates at a density of 5 x 104 cells/well one day prior to transfection. The medium was replaced with antibiotic-free 10% FBS DMEM medium before transfection. Transfection was carried out with 25 ng/well of Gal4-RORα-LBD, Gal4-RORγ-LBD, Gal4-mut-RORα-LBD, or Gal4-mut-RORγ-LBD, 25 ng/well of pG5-Luc, and 5 ng/well of renilla for 8 hours. Test compounds were added after transfection, and luminescence signal was detected after 48 hours using the Luciferase dual reporter gene assay kit. All liquids were transferred to a white light-proof 96-well plate. The DLR kit was used to detect luciferase expression, and luminescence was measured by a Biotek Synergyh1 multifunctional microplate reader. Data were analyzed as relative luciferase unit (RLU) based on firefly luciferase activity (fluc) and renilla luciferase (rluc) activity in three parallel groups.

For CCK8 analysis, cells were seeded in 96-well culture plates at a density of 1×104 cells per well with three replicate wells for each group. After overnight culture, cells were treated with drug-containing medium at different concentrations (0, 1, 2, 4, and 8 μmol/L) for 48 hours before performing a CCK-8 assay to calculate the IC50. CCK8 analysis was repeated three times to observe cell viability after drug treatment.

Data were analyzed using SPSS26.0 statistical software, and results were presented as mean ± S.D. Paired t-tests (2-tailed) were used to compare data from different drug groups at the same time point, while one-way Anova and Fisher's LSD were used to analyze the same drug at different time points. P-values < 0.05 were considered statistically significant.