MLO-Y4 cells were plated in type I rat tail collagen-coated six-well plates (Biocoat, Becton Dickinson) or coverslips and maintained in α–minimal essential medium (α-MEM; Gibco) supplemented with 5% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin (P/S) (Sigma-Aldrich). For transwell culture, MLO-Y4 ρ° cells were plated on the outer side of the transwell membrane. After 24 hours, MLO-Y4 ρ° cells, MLO-Y4 parental cells, primary osteocytes, or si-Mfn2 MLO-Y4 cells were plated on the inner side of the transwell membrane and cultured for up to 4 days. The cells were maintained at 37°C in a 5% CO2 humidified incubator.

For ex vivo culture, calvariae from 10-day-old mice were washed three times with phosphate-buffered solution (PBS) and three times with α-MEM containing 1% P/S. The periosteum was removed from the calvariae with scraping and extensive washing. Calvariae were maintained in α-MEM supplemented with 10% FBS and 1% P/S at 37°C and 5% CO2 in a humidified incubator. Ex vivo cultured calvariae were fixed in 4% paraformaldehyde (PFA) for immunostaining and maintained in culture medium in 35-mm glass-bottom petri dishes (P35g-1.5-14-C, MatTek) for subsequent confocal imaging. MLO-Y4 ρ° cells were generated by culturing parental MLO-Y4 for 15 weeks in α-MEM medium supplemented with EtBr (50 ng/ml), pyruvate (110 μg/ml), and uridine (50 μg/ml) and subsequently transferred to the medium lacking EtBr.

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