Mccoy s 5a medium

The human bladder cancer cell line T24 used in this research was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in McCoy’s 5A medium (iCell, China) containing 10% fetal bovine serum (FBS, BI, ISR). The cells were grown at 37 °C and 5% CO₂. The human TGF beta 1 (TGF-β1) protein was purchased from MedChemExpress (HY-P7118). T24 cells were treated with TGF-β1 (10 ng/ml) for 48 h.

Human normal colon epithelium cell lines (NCM460) and human colorectal cancer cell lines (SW480 and HCT116) were bought from the American Type Culture Collection (ATCC, USA). Human colorectal cancer cell lines (HT29 and RKO) were purchased from the iCell (Shanghai, China). SW480 was grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, USA). NCM460 and LOVO were grown in RPMI (1640) medium (GIBCO, USA). RKO was grown in modified Eagle's medium (MEM) (GIBCO, USA). HT29 was grown in McCOY's 5A medium (iCell, China). All cells were cultured in a constant temperature and humidified incubator at 37°C with 5% CO₂.

The human renal cortex proximal tubule epithelial cells (HK-2 cells) were cultured in DMEM/F12 medium (Gibco, USA), the human primary clear cell adenocarcinoma cells (7860 cells) and the human skin metastasis-derived clear cell renal cell carcinoma cells (Caki-1 cells) were maintained in RPMI 1640 medium (Gibco, USA) and McCoy’s 5A Medium (iCell, China), respectively. All mediums were supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL of penicillin and 100 U/mL of streptomycin (Gibco, USA). The conditions of cell cultures were 37°C and 5% CO2.
Using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, China) to extract the total RNA following the manufacturer’s instructions. Then, the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China) was used to perform the reverse transcription reactions and the AceQ Universal SYBR qPCR Master Mix (Vazyme, China) was used to complete qPCR.
The comparative Ct method was used to analyze the relative expression levels of four FRGs: 2−ΔΔCt (ΔΔCt = (ΔCt of FRG) − (ΔCt of GAPDH). GAPDH was used as the internal normalized reference to FRGs. The specific primer sequences used were listed in Table 1.