HK-2 cells (ATCC, Manassas, VA, USA), which were immortalized by transduction with human papillomavirus 16 E6/E7 genes, were maintained in a keratinocyte serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5 ng/mL human recombinant epidermal growth factor 1–53 and 50 μg/mL bovine pituitary extract at 37 °C in a humidified 5% CO2.
For analyzing the protective role of SGLT2 inhibitors in cisplatin-induced cytotoxicity, HK-2 cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin (Sigma-Aldrich, St. Louis, MO, USA) in the absence or presence of 1–25 μM canagliflozin, dapagliflozin, or empagliflozin (APExBIO, Houston, TX, USA) for 24 h.
To test whether canagliflozin induces autophagy or modulates AMPK-mTOR pathway and whether the protective effect of canagliflozin on cisplatin-induced cytotoxicity occurs in autophagy or AMPK activation-dependent manner, the cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin in the absence or presence of 10 μM canagliflozin and/or 10 μM chloroquine, 5 nM bafilomycin A (Sigma-Aldrich), or 1 μM compound C (APExBIO) for 24 h.
For analyzing the protective role of SGLT2 inhibitors in cisplatin-induced cytotoxicity, HK-2 cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin (Sigma-Aldrich, St. Louis, MO, USA) in the absence or presence of 1–25 μM canagliflozin, dapagliflozin, or empagliflozin (APExBIO, Houston, TX, USA) for 24 h.
To test whether canagliflozin induces autophagy or modulates AMPK-mTOR pathway and whether the protective effect of canagliflozin on cisplatin-induced cytotoxicity occurs in autophagy or AMPK activation-dependent manner, the cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin in the absence or presence of 10 μM canagliflozin and/or 10 μM chloroquine, 5 nM bafilomycin A (Sigma-Aldrich), or 1 μM compound C (APExBIO) for 24 h.