In vivo xenograft model

All experimental procedures were conducted following a protocol approved by the Institutional Animal Care and Use Committee of Asan Institute for Life Sciences (Permit Number: 2015-12-087, Seoul, Korea). All efforts to minimize suffering were made in this experiment. To establish a xenograft model, male nude mice (6∼8 weeks of age) were purchased from Charles River Laboratories (Wilmington, MA). Tumors were grown by implanting cells (1 × 10⁶ cells/0.1 ml) in 50% matrigel (BD Biosciences, San Jose, CA) and were subcutaneously injected into the right flank of each animal. When the tumor volume reached approximately 100 mm³, the mice were divided into four treatment groups (n = 5 per group): untreated, TRAIL (10 mg/kg), BNTX (0.5 mg/kg), and combination of BNTX (0.5 mg/kg) and TRAIL (10 mg/kg). Both TRAIL and BNTX were dissolved in a HEPES buffer. All drugs were administered by an intraperitoneal injection twice a week. To assess tumor size, the length (L) and width (W) of each tumor was measured using calipers, and the tumor volume (TV) was calculated as TV = (L × W²)/2. Lysates were prepared from tumor tissues, and protein levels were detected by western blotting using anti-XIAP, anti-cleaved caspase-3 and anti-PARP antibodies.