Nexavar

For cell growth assessment, HCC cells were seeded at 7500 cells per well in 96-well plates 24 h after transfection. Cell viability was measured at the indicated times using Cell Counting Kit-8 (Dojindo) according to the manufacturer’s instructions (WST-8 assay). The absorbance at 450 nm was measured using a Synergy H4 Microplate Reader system (BioTek). For the evaluation of sorafenib cytotoxicity, miR-493-5p mimic and control mimic-transfected cells were seeded in 96-well plates (10,000 cells/well). The next day, the medium was changed, and the cells were cultured in medium containing different concentrations of sorafenib for 48 h. Cell viability was measured as mentioned above. Sorafenib (Nexavar; PubChem CID: 216239) was purchased from Santa Cruz Biotechnology (#SC-220125).

The human proximal tubular epithelial cells HK-2 (wild type p53), the human renal cell carcinoma RCC-Shaw (ccRCC-derived cell line with wt-p53), the human renal carcinoma of BHD (Birt-Hogg-Dubè) origin UOK-257 cells (RCC cell line with mutated p53) and the colon cancer cell line HCT116 were cultured in Dulbecco’s modified Eagle’s medium (D-MEM) plus 10% foetal bovine serum (FBS), L-Glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37 °C, 5% CO₂. Nutlin-3 10 μM (Cayman), Cisplatin 7.5 μM (Sigma), Sorafenib 10 μM (SantaCruz) and Axitinib 10 μM (SantaCruz) were used for 24 h [2 (link), 7 (link), 21 (link)].
In particular, Sorafenib and Axitinib are normally used for treatment of ccRCC patients and are commercially available with the name Nexavar^® and Inlyta^®, respectively.