Male c57bl 6 mice

C57BL/6 male mice were purchased from KOATECH (Seoul, Korea) and kept per cage in a temperature-controlled room under a 12 h light/12 h dark cycle. Animals were provided with standard rodent chow containing 6.2% (wt/wt) total fat (Envigo, Indianapolis, IN, USA) and water. All experiments conformed to the guidelines and regulations issued by the Animal Care Committee of our institute, and all animal experiments were approved by the Institutional Animal Care and Use Committees of the Yeungnam University (YUMC-AEC2017–013 and YUMC-ACE2019–033). The acute skeletal muscle injury model was generated by a single intramuscular (left gastrocnemius, 100 μl 10 uM) injection of cardiotoxin (CTX; Najamossambica; Sigma Aldrich, St. Louis, MO, USA). Contralateral right gastrocnemius muscles were injected with phosphate buffered saline and used as controls. To investigate the effects of the CWE of G. uralensis or liquiritigenin on muscle regeneration, two-month-old mice were orally administered CWE of G. uralensis (100 mg/kg) or liquiritigenin (15 mg/kg) once a day for 9 days from one day before CTX injection. Seven days after CTX injection, mice were anesthetized with avertin i.p. (Sigma Aldrich, a mixture of tert-amyl alcohol and 2,2,2-tribromoethanol) and gastrocnemius muscles were excised for Western blotting and immunohistochemistry analysis.

For this study, 7-week-old C57BL/6 male mice bought from KOATECH (Gyeonggi, Korea) were randomly assigned into cages with four mice in each of them in a room with 12-h light cycle (lights on at 6:00 am and off at 6:00 pm) at 22 ± 2 °C and 55 ± 5 of humidity and with water and feed freely supplied for 1-week prior experiment.
All protocols for animal experiments were approved by the Institutional Animal Care and Committee (IACUC) of Myongji University (MJIACUC-2021003) and thus conducted in accordance with the NIH guide for the care and Use of Laboratory Animals.

Five-week-old C57BL/6 male mice were purchased from KOATECH (Korea). Animal care and experiments were carried out according to the guidelines of the Korean Food and Drug Administration, and protocols were reviewed and approved by the Institutional Review Board of Yonsei University. Animals were fed standard rodent chow and water and maintained under a 12 hours light/12 hours dark cycle at 22–25°C with a relative humidity of 45–55%. The shCon-GFP and shSur8-GFP B16-F10 cells were trypsinized and washed in PBS. Cells (1 × 10⁶) were suspended in 150 μl PBS and injected intravenously into the tail vein of 6-week-old mice. The mice were anesthesized after 21 days, and the lungs were isolated, photographed, and fixed in 4% PFA for 3 days at 4°C. Metastasized tumor nodules on the lung surface were counted after fixation.

To assess the effects of the PKCδ inhibitors on calvarial bone remodelling, 8-week-old C57BL/6 male mice purchased from Koatech, Inc. were maintained in the mouse facility. All procedures using the mice were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Yeungnam University College of Medicine and were approved by the Institutional Animal Research Review Committee of Yeungnam University College of Medicine (permission number YUMC-AEC2016-038). Rottlerin (2 mg/kg), delta PKC (8–17) (5 mg/kg), or PBS (control) was injected onto the calvarial periosteum of the mice every day for 5 days, and then the mice were sacrificed on day 3 after the final injection. Calvarial specimens were surgically dissected from the mice, fixed in 3.7% formaldehyde, decalcified with EDTA solution, and sectioned using a microtome. The sections were then stained with TRAP to detect the osteoclasts on the calvarial bone surface and with haematoxylin and eosin (H&E) to visualise the bone marrow cavity, to assess osteoclastic bone-resorbing activity. The number of TPAP-positive osteoclasts was counted under a light microscope. To measure the area of the bone marrow cavity, which reflects osteoclastic bone-resorbing activity, images were scanned using an Aperio ScanScope (Model T3) and analysed using ImageScope software, version 6 (Aperio Technologies, Vista, CA, USA).

Osteoclast precursors were isolated from the tibia and femur of 6-week-old C57BL/6 male mice (Koatech, Inc., Gyeonggido, Korea) by flushing the bone marrow as previously described^{29 (link)}. In brief, erythrocytes within the bone marrow fraction were lysed with red blood cell lysis buffer (Sigma-Aldrich). The remaining cells were cultured in alpha minimum essential medium (α-MEM; Hyclone, Logan, UT, USA) containing 10% foetal bovine serum (FBS, Hyclone), 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and recombinant human M-CSF (5 ng/ml) for 12 h at 37 °C in 5% CO₂. To generate osteoclast precursors, the floating cells were further cultured in α-MEM containing M-CSF (30 ng/ml) for 3 days. For osteoclast differentiation, osteoclast precursors (2.5 × 10⁴ cells per well) were seeded onto 48-well culture plates and then differentiated into osteoclasts in the presence of M-CSF (30 ng/ml) and recombinant mouse RANKL (100 ng/ml) for 4 days. The medium was exchanged on day 2. To evaluate osteoclast differentiation, cells were stained for tartrate-resistant acid phosphatase (TRAP) using the Leukocyte Acid Phosphatase Staining Kit (Sigma-Aldrich) according to the manufacturer’s instructions. TRAP-positive multinucleated cells (TRAP⁺ MNCs) with more than three nuclei were counted under a light microscope.

Cxxc5^−/− mice were established in a previous study (Kim et al, 2015 (link)). To manipulate growth plate senescence by estrogen, 3-wk-old Cxxc5^+/+ and Cxxc5^−/− male mice received weekly i.m. injections of either 70 μg/kg estradiol (E₂) cypionate (Sigma-Aldrich) or vehicle (cottonseed oil) for 3 wk. To investigate the effects of KY19382 treatment on longitudinal bone growth, C57BL/6 male mice were purchased from KOATECH (Gyeonggido, Korea). KY19382 (0.1 mg/kg) was administered daily by i.p. injection to 3- and 7-wk-old mice for 2 wk or to 3-wk-old mice for 10 wk. For BrdU labeling experiments, the mice were i.p. injected with 50 mg/kg BrdU (Sigma-Aldrich) before 24 h to euthanize. All animal procedures were approved by the Institutional Animal Care and Use Committee of Yonsei University (Korea) and conducted based on the guidelines of the Korean Food and Drug Administration.