Human Renal Epithelial Cell Line 293T: Transfection Protocol and CCK8 Analysis for Drug Viability Studies

Human renal epithelial cell line 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). They were then inoculated into 96-well plates at 5 x 104 cells/well 1 day prior to transfection. Prior to transfection, the medium was replaced with antibiotic-free 10% fetal bovine serum DMEM medium. Co-transfection of 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 was done for 8 hours, after which 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 Dual-luciferase reporter (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 was calculated as Firefly luciferase activity (fluc) / renilla luciferase (rluc) activity.

For CCK8 analysis, cells were inoculated in 96-well culture plates at 1×104 cells per well, with 3 replicate wells designed for each group. After the cells were cultured overnight, they were changed to drug-containing medium with a drug concentration gradient of 0, 1, 2, 4 and 8 μmol/L. Cells were incubated at 37°C in a 5% CO2 incubator for 48 hours before CCK-8 assay was performed and IC50 was calculated. 10 μL of CCK8 was added to each well and incubated at 37°C in a 5% CO2 incubator for 2 hours. OD values at 450 nm were measured on an enzyme marker and data were analyzed. CCK8 analysis was used to observe the viability of cells after drug treatment. CCK8 analysis was repeated three times and the half lethal dose (IC50) of CSL was calculated.

Data were analyzed using SPSS26.0 statistical software, with data presented as mean ± S.D. Paired t-tests (2-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 time. A significance level of P < 0.05 was used to determine significant differences.