A498 cells

Human renal cancer A498 and ACHN cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were maintained under standard conditions (5% CO2, 37°C, and 95% humidity). A498 cells were cultured in RPMI-1640 medium (GIBCO, Grand Island, New York, USA), and ACHN cells were cultured in DMEM (GIBCO) containing 10% heat-inactivated fetal bovine serum (Ab frontier, Korea) and 1% penicillin/streptomycin (GIBCO). Human embryonic kidney 293 T cells were purchased from American type culture collection (ATCC, Rockville, MD). The cells were maintained under standard conditions (5% CO2, 37°C, and 95% humidity). The cells were cultured in DMEM media (GIBCO) containing 10% heat-inactivated fetal bovine serum (Ab frontier, Korea) and 1% penicillin/streptomycin (GIBCO).

The ccRCC A-498 cells and 786-O were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). A-498 cells and 786-O cells were, respectively, cultured in MEM Alpha medium (Gibco by Life Technologies, Grand Island, NY, USA) and PRMI 1640 (Gibco by Life Technologies) containing 10% fetal bovine serum (BI, Kibbutz Beit Haemek, Israel) and 0.1 mg/mL streptomycin (BBI Life Sciences, Shanghai, China) and 100 U/mL penicillin at 37°C in a humidified incubator with 5% CO₂. The sequence of shRNA targeting UBE2I was cloned into a pLVX vector. The shRNA sequences were as follows: 5′-CCGG GAA​CTT​CTA​AAT​GAA​CCA​AAT​CTC​GAG​ATT​TGG​TTC​ATT​TAG​AAG​TTC​TTT​TTG-3′. The transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines.