Generation of Nude Mouse Model of Xenograft Tumor

Male BALB/c athymic nude mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl; age, 5 wk; N = 5/group) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and bred in the Animal Center of the College of Medicine, National Taiwan University. Mice were housed in ventilated caging units under positive pressure in a specific pathogen-free facility with a standard 12-hour light-dark cycle and free access to mouse chow (LabDiet 5053; Purina Mills, Richmond, IN) and sterile water. These mice were adapted for 1 week before xenograft implantation. Huh-7 cells (1 × 10⁶ cells/mouse) were transfected 3 times with siCtrl and siRBMY, as well as transduced twice with shCtrl, shRBMY, and shRBMY+HA-RBMY, and were resuspended in 150 μL FBS-free DMEM/F12 (Thermo Fisher Scientific) plus Matrigel (BD Biosciences, San Jose, CA) in a 1:1 ratio. Cells were inoculated subcutaneously into the right and left flanks of nude mice. Mice were monitored for the growth of implanted cells and killed after 90 days. All animal procedures were in compliance and approved by the Institutional Animal Care and Use Committee of National Taiwan University, College of Medicine (Institutional Animal Care and Use Committee approval 20150409).