BMMs were cultured in the presence of macrophage colony-stimulating factor (M-CSF) (20 ng/ml) and RANKL (20 ng/ml) for 5 days, and TRAP staining and the pit formation assay were performed as previously described (63, 64). Cells were treated with BMS-345541 (Abcam) at 0.16 μM or Akt inhibitor X (Cayman) at 0.2 μM followed by TRAP staining. Cells were cultured with 10 μM BrdU for 45 min and were prepared for analysis of BrdU incorporation using the FITC BrdU Flow Kit (BD Biosciences) by flow cytometric analysis using the FACSVerse (BD Biosciences). Cell death assay was performed with FITC–Annexin V (BD Biosciences) and propidium iodide by FACSVerse. Data were analyzed by FACSuite software (BD Biosciences).

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