Characterization of RCC Cell Lines

The RCC cell lines ACHN, Caki1, and A498 were obtained from American Type Culture Collection (Manassas, VA, USA). ACHN is derived from pleural effusion in metastatic RCC having wild type of VHL [23 (link), 24 (link)]. Caki1 and A498 cells come from clear cell RCC with VHL wild type [23 (link), 25 (link)], and clear cell RCC with VHL mutation (426_429delTGAC) [25 (link)], respectively. Cells were cultured in RPMI medium supplemented with 50 μg/mL of kanamycin and 10 % fetal bovine serum in an incubator at 5 % CO₂ and 37 °C. Human renal proximal tubular epithelial cell (HRPTEpC) was obtained from Cell applications Inc (San Diego, CA, USA). Cells were cultured in RenaEpi cell growth medium with growth supplements in an incubator at 5 % CO₂ and 37 °C. AR-A014418 was purchased from Calbiochem (San Diego, CA, USA). Two other GSK-3 inhibitors, SB-216763 and TDZD8, were obtained from Cayman Chemicals (Ann Arbor, MI, USA) and Sigma-Aldrich Japan (Tokyo, Japan), respectively. Rapamycin and everolimus were obtained from Selleck Chemicals (Houston, TX, USA), LY294002 was from Wako Pure Chemical Industries (Tokyo, Japan), recombinant GSK-3β was purchased from New England Biolabs (NEB) Japan (Tokyo, Japan), and recombinant GST-4EBP1 was obtained from Sigma-Aldrich Japan.