Human Renal Epithelial Cell Line 293T Transfection and CCK8 Analysis for Drug Efficacy Evaluation

Human renal epithelial cell line 293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and inoculated into 96-well plates at a density of 5 × 104 cells/well one day prior to transfection. Prior to transfection, the medium was replaced with antibiotic-free 10% FBS DMEM medium. The cells were co-transfected 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. After 8 hours of co-transfection, the test compounds were added. After 48 hours of incubation, the luminescence signal was detected using the Luciferase dual reporter gene assay kit. All liquids in the 96-well plate were transferred to a white light-proof 96-well plate. The DLR kit was used to detect the expression of luciferase in cells. Luminescence was detected by a Biotek Synergyh1 multifunctional microplate reader, and data were statistically analyzed as relative luciferase unit (RLU) (3 parallel groups). RLU = Firefly luciferase activity (fluc) / renilla luciferase (rluc) activity.

For CCK8 analysis, cells were inoculated in 96-well culture plates at a density of 1 × 104 cells per well, with 3 replicate wells designed for each group. After overnight culture, the cells were treated with drug-containing medium (drug concentration gradient of 0, 1, 2, 4 and 8 μmol/L). The cells were incubated at 37°C in a 5% CO2 incubator for 48 hours before the CCK-8 assay was performed and IC50 was calculated. Ten microliters of CCK8 was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 2 hours. The OD values at 450 nm were measured using an enzyme marker, and data were analyzed. CCK8 analysis was repeated three times, and the half lethal dose (IC50) of CSL was calculated.

Statistical analysis was performed using SPSS26.0 statistical software, with data presented as mean ± S.D. Paired t-tests (two-tailed) were used between data from different drug groups at the same time point. One-way ANOVA and Fisher's LSD were used to analyze the data of the same drug at different times. A p-value of less than 0.05 was considered significant.