Tamoxifen

Tamoxifen (Toronto Research Chemical, T006000) was dissolved in corn oil at a concentration of 50 mg/mL. Unless otherwise noted, all experimental mice were between 8 and 13 weeks of age and were administered Tamoxifen at a dose of 10 mg via oral gavage for 4 or 5 consecutive days. Muscle injury was induced at least 3 days after the last Tamoxifen dose by injecting 100 μL of 10 μM Naja mossambica mossambica cardiotoxin (Sigma-Aldrich, C9759) in PBS into the left TA or gastrocnemius muscle.

All surgeries were performed on 8-week-old male mice under general anesthesia with the aid of an operating microscope. The mice were anesthetized by intraperitoneal injection with a mixture of medetomidine (0.3 mg/kg; #0002949, Nippon Zenyaku Kogyo, Koriyama, Japan), midazolam (4.0 mg/kg; #169738, Astellas, Tokyo, Japan), and butorphanol (5.0 mg/kg; #016440, Meiji Seika Pharma, Tokyo, Japan). A 27-gauge needle was used for the puncture, which was performed percutaneously from the outside of the Achilles tendon body, and this process was repeated five times at different sites on the Achilles tendon body of each mouse, as previously described (13 (link)). In the sham operation, the needle was inserted through the skin without touching the Achilles tendon (13 (link)). For tamoxifen induction, mice were injected intraperitoneally with tamoxifen (10 mg/ml; #T006000, Toronto Research Chemicals, Toronto, Canada) in corn oil at a dose of 50 μg/g body weight. The injection was performed twice over consecutive days before or after ATP induction. For the administration of an antibody against RSPO2, ATP was induced in 8-week-old WT mice, which then received 10 μl of RSPO2 antibody solution or vehicle (10 μg/ml) [phosphate-buffered saline (PBS)] injected into the Achilles tendon every day for 20 days starting from the day after ATP induction.