Balb c nude mice nu nu

Five-week-old male BALB/c nude mice (nu/nu) were purchased from Orient Bio Inc. (Gyeonggi-do, Korea). Experiments on animals were performed in accordance with the Guidelines for the Care and Use of Animals of the Kongju National University Animals Care Committee (Chungcheongnam-do, Korea). Mice were maintained under a 12 h light/dark cycle, and housed under controlled temperature (23 ± 3 °C) and humidity (40% ± 10%) conditions. Mice were allowed access to laboratory pelleted food and water ad libitum. DU-145 cells were maintained in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. DU-145 cells were harvested from cultures by exposure to 0.25% trypsin. Trypsinization was stopped with a solution containing 10% FBS, and cells were then washed twice and resuspended in RPMI-1640 medium. A total of 2 × 10⁷ cells in 0.2 mL of medium were injected subcutaneously into the right flank of donor nude mice. Seven days after the subcutaneous injection, DU-145 cells growing under the skin of nude mice established tumors. All animal experiments were performed following the approval of the Institutional Animal Care and Use Committee according to the guidelines of Kongju National University (KNU_2015-06).

For animal study, five-week-old, female, athymic BALB/c nude mice (nu/nu) were purchased from OrientBio, Inc. (Daejeon, Korea) and maintained for 1 week with free access to sterile standard mouse chow (NIH-7 open formula) and water before use. Mice were housed randomly at 50% ± 20% humidity and approximately 21 ± 2 °C on a 12 h light–dark cycle (n = 5 mice/group). All animal experimental procedures were performed according to the National Institutes of Health guidelines and a protocol approved by the Institutional Animal Care and Use Committee of Kyung Hee University.

Five-week-old female, athymic BALB/c nude mice (nu/nu) were purchased from OrientBio, Inc. (Daejeon, Republic of Korea), and housed in a pathogen-free room for one week with NIH-7 open formula. The mice were divided randomly into three groups. All animal studies were performed according to the guidelines of the National Institutes of Health and Kyung-Hee University Animal Care and Use Committee. For the xenograft mice experiment, 1 × 10⁷ MDAMB231 cells were mixed in PBS, and injected subcutaneously (sc) into the right dorsal flank of nude mice. When the tumor volume reached approximately 200 mm³, the mice were randomly grouped (n = 10 per group). Nodakenin (10 or 30 mg/kg) was then administered intraperitoneally (ip) twice weekly. Tumor volume was calculated using the following formula: (L × W²)/2 (mm³).