Wst 1 kit

AZD1775, olaparib, and gemcitabine (purchased from Selleck Chemicals, Houston, TX, USA) were resuspended in Dimethyl sulfoxide (DMSO). At a concentration of 1 × 10⁴ cells/well, KLE and OVCAR3 cells were seeded in conventional 96-well plates with 100 μL of medium and allowed to adhere overnight. Cells were then exposed the following day to drug treatments for 72 h, and cell proliferation was analyzed using a WST-1 kit (Sigma, St. Louis, MO, USA). The WST-1 assay is a colorimetric test based on cleavage of the tetrazolium salt WST-1 into orange formazan by mitochondrial dehydrogenases in viable cells. The result was reported as a percentage of viable cells in the treatment arms compared to the control arm of each experiment. Each condition was performed in three replicated wells, and the data represent three independent experiments.

Kidney cancer cell lines 786-O and A498 were purchased from ATCC. Ren-02, RCC4 and HEK293T were described previously (Niu et al., 2012 (link); Negrotto et al., 2011 (link)). UMRC2 and UMRC6 cells were a gift from Dr. Qing Zhang from University of North Carolina at Chapel Hill. All cell lines were maintained in 37°C incubator with 5% CO₂ in glutamine-containing DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Mycoplasma contamination is tested with a PCR-based kit. The cells will be treated with antibiotics to eradicate the mycoplasma if it is detected. The cell proliferation rate was measured with a WST-1 kit from Sigma Aldrich according to the manufacturer’s instruction.
The packaging of shRNA viral particles, the infection and selection, and the expression of shRNA resistant plasmid were the same as described before (Kondo et al., 2002 (link)).