Balb c nu nu female mice

Female Balb/c nu-nu mice (5 weeks old, Japan SLC, Inc., Shizuoka, Japan) were housed under a 12-h light/12-h dark cycle and given free access to food and water. HT1080 cells (5×10⁶ cells/100 µl PBS (−)/mouse) were subcutaneously inoculated into the right hind leg of Balb/c nu-nu mice. Animals were used for experiments two weeks after inoculation when the mean tumor size was 5.7±2.2 mm along the major axis.

Female BALB/c nu/nu mice aged 7–9 weeks (Japan SLC, Hamamatsu, Japan) were used for xenograft tumor formation. The 1 × 10⁶ HeLa cells stably expressing yellow Nano-lantern and cyan Nano-lantern in 50 μl GelTrex/PBS (1:1) (ThermoFisher Scientific) were subcutaneously injected on the left and right flanks of the mice. Mice were imaged two weeks after transplantation. For the tumor metastasis study, mice were intravenously injected with 1 × 10⁵ 4T1 murine breast tumor cells stably expressing hyBRET-ERK or hyBRET-ERK-TA in PBS in the tail vein and analyzed 2–3 weeks after the 4T1 tumor injection. Transgenic mice were generated by Tol2-mediated gene transfer^{54 (link)}. Briefly, fertilized eggs derived from Jcl:B6C3F1 (B57BL/6N Jcl X C3H/HeN Jcl) mice were microinjected with a mixture of Tol2 mRNA and the pT2ADW- hyBRET-ERK. Founder animals were bred with Jcl:ICR mice to produce stable lines. Newborn mice were illuminated with a blue flashlight LEDGFP-3W (Optocode, Tokyo) and inspected for green or red fluorescence through yellow-colored glasses. The animal protocols were reviewed and approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (No. 10584, 14079, 15064, 16038, and 17539) and the methods were carried out in accordance with the relevant guidelines and regulations.

Mice were used for tumor implantation studies, and rats were used for MRI scanning, biodistribution analysis, and toxicity testing because their body size and weight were more suitable for these studies.
Female balb/c nu/nu mice (17–23 g) (Japan SLC, Inc., Shizuoka, Japan), 6 weeks old, were anesthetized with isoflurane together with intraperitoneal injection of tribromoethanol (250 mg/kg). In the case of brain injection, a burr hole was made in the skull 0.5 mm anterior and 2.0 mm lateral to the bregma before tumor cells were stereotactically injected by a 30-gauge injection canula to a depth of 4.0 mm. The injection volume was 10 μl per body31 (link)32 (link).
Crl: CD (SD) male rats (190–299 g), 8 weeks old, were purchased from Charles River Laboratories International, Inc. (USA). Under general anesthesia, a burr hole was made in the skull 0.2–1.0 mm anterior and 3.0 mm lateral to the bregma, and a canula was inserted to 5.0 mm depth from the outer skull33 (link). Fe(Salen) (0.12–0.60 mg/body) was injected into the brain. The injection volume was 10–20 μl per body.