Muscle Incubation with Undercarboxylated Osteocalcin

Muscles were evenly divided longitudinally into halves to improve the effusion of ucOC into muscle fiber ex vivo, similar to what has been performed in rat muscle in previous studies (16 (link)–18 (link)). The whole ucOC stimulation process is shown in Figure S1 in Supplementary Material. In experiments without the ERK inhibitor U0126, muscle samples were preincubated in 30°C baths containing carbogenated KHB buffer for 1 h. In experiments with U0126 (N = 5), after 30 min preincubation, muscle samples were exposed to the ERK inhibitor U0126 (1 µM) (Cell Signaling, MA, USA) or dimethyl sulfoxide (DMSO) vehicle (Sigma-Aldrich, MO, USA) for 30 min. Then, muscle samples were stimulated for 90 min with increasing doses [0 ng mL⁻¹ (N = 6), 0.3 ng mL⁻¹ (N = 10), 3 ng mL⁻¹ (N = 10), 10 ng mL⁻¹ (N = 14), or 30 ng mL⁻¹ (N = 10)] of recombinant ucOC (Bachem, Bubendorf, Switzerland). These doses of ucOC were chosen because they are within the physiological range in mice (7 (link), 31 (link)). In experiments without U0126, muscle halves from the same mouse were treated with KHB buffer control or ucOC. In experiments with U0126, muscle halves from the same mouse were treated with DMSO, DMSO with ucOC, U0126, and U0126 with ucOC, respectively.