Balb c nude mice

All animals were cared for and experiments were performed using protocols approved by the institutional review board and the institutional animal care and use committee of Keio University. MCF7 cells expressing activated H-Ras^V12-expressing lentivirus and either tet-BTG2 or tet-LacZ were generated using the above protocols. Xenografts were established by injecting 5 × 10⁶ cells in 100 μL of phosphate-buffered saline into the mammary fat pad of 6-week-old female, BALB/c, nude mice (Sankyo Labo Service, Tokyo, Japan). Each group consisted of 8–11 mice. Tumor volumes were measured once every two days using the formula length × (width)². To determine the effect of tamoxifen on tumor growth, these tumor-bearing mice were administered water containing tetracycline or tamoxifen pellets (21-day time-release containing 1.5 mg tamoxifen; Innovative Research of America, Sarasota, FL, USA) from the day when the xenografted tumor grew to be over 125 mm³ and then killed after 3 weeks of treatment.

TGS‐01 cells expressing luciferase were transduced with adenovirus to express LacZ (ad‐LacZ), EPHA6 WT (ad‐EPHA6‐WT), or EPHA6 kinase‐dead mutant (ad‐EPHA6‐KD) (MOI 60) together with pretreatment with 200 ng/mL BMP‐2 for 2 days prior to intracranial injection in 6‐week‐old female BALB/c nude mice (Sankyo Labo Service). A total of 75 000 cells in 3 μL medium was stereotactically injected over a 1‐minute period. Injection coordinates, imaging, and killing methods were conducted as previously described.9, 27 Log‐rank test was used for statistical analysis.

Four-week-old female BALB/c nude mice were purchased from Sankyo Labo Service corporation, Inc. The animals had free access to a commercial diet and water in animal cages under a pathogen-free condition at 25±1 °C and humidity of 45±10% in a 12 h dark and light cycles.
In order to test the antitumor activity of fullerene derivatives in vivo, A549 cells (1.0×10⁷ cells) were injected subcutaneously into female BALB/c nude mice aged 4 weeks. After injection of A549 cells, the mice were treated daily for 6 days with an intraperitoneal injection of fullerene derivatives. The animals were sacrificed and the weights of the tumor, liver and spleen were recorded at two weeks after tumor cell inoculation (Day 20) (n=4).
Animal welfare and experimental procedures were performed strictly in accordance with the Care and Use of Laboratory Animals and approved by the Keio University Animal Research Committee (approval number: 17043). All efforts were made to minimize the animals’ suffering and to reduce the number of animals used.