Dmem medium

ccRCC tissues and adjacent normal tissues were obtained from patients undergoing urological surgery in Wuhan University People’s Hospital. The privacy rights of human subjects always be observed. Human ccRCC cell lines 786-O, 769P, A498, ACHN and normal kidney tubular epithelial cell HK-2 were obtained from the ATCC (American Type Culture Collection). HK-2 was cultivated in DMEM medium (Cytiva, Logan Utah, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY, USA) and ccRCC cells were cultured in RPMI 1640 medium (Cytiva, Logan Utah, USA) supplemented with 10% FBS. All cells were cultured in the same humidified atmosphere (37 °C with 5% CO2). 786-O cell line was transfected by NR1H4-specific siRNA for 6 h. Meanwhile, nontargeting siRNA (Sangon Biotech, Shanghai, China) was used to transfect 786-O cells as a negative control. The siRNA transfections were performed using Lipo6000 transfection reagent (Beyotime, Shanghai, China), according to the manufacturer’s protocol. After 48 h, cell experiments were performed.

3T3-L1 cells (ATCC) were plated at 50,000 cells per well in a 24 well cell culture plate (Corning Incorporated, Kennebunk, ME, USA) for 24 h until confluent. DMEM medium (Cytiva, Logan, UT, USA) was replaced, and cells were incubated with microparticles in DMEM for 24 h at 0, 10, 20, 50, 100, and 200 µL/mL concentrations with three replicates per concentration. Medium was removed and replaced with 250 µL of fresh medium and 50 µL of MTT stock solution (Life Technologies Corporation, Eugene, OR, USA) to incubate at 37 °C for 4 h. Three additional wells of medium and MTT stock solution were incubated without cells as a control. At the end of the incubation, all medium was removed, and 1 mL of DMSO (Sigma Aldrich, St. Louis, MO, USA) was added to each well and incubated at 37 °C for 10 min. The contents of the wells were collected, centrifuged to remove debris, and the DMSO supernatant was plated in a 96 well plate (Thermo Scientific, Rochester, NY, USA) with three replicates per sample and read in the ELx800 plate reader (BioTek, Winooski, VT, USA) for absorbance at 540 nm.

A human hepatic carcinoma (HepG2) cell line was purchased from the Shanghai Cell Bank of Type Culture Collection of China. Cells were cultured continuously at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM) medium (Cytiva, Results Way, Marlborough, MA, USA) with 10% fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). To verify the effect of curcumin on the detoxification effects of different heavy metals, we treated curcumin at non-toxic concentrations (5, 10 and 20 µM). The stock solution of curcumin and heavy metals were diluted with DMEM to the concentration of the working solution and then exposed to cells.