PCI-32765 blocked Btk-mediating osteoclastogenic signaling pathway and impacted osteoclastogenesis. (A) CD14⁺ OC precursor cells (OCPs) from normal human donor or mouse raw267.4 cells were stimulated with RANKL/M-CSF for the indicated time intervals. Cell lysates were subjected to immunoblotting with antiphosphotyrosine antibodies. Anti–α-tubulin and -Btk mAbs served as loading controls. (B) OCPs were pretreated with (+) or without (−) PCI-32765 (100nM) for 2 hours before stimulation with RANKL/M-CSF. (C) TRAP staining was performed at 10 days of OC culture to identify mature OCs (> 3 nuclei, > 50 μm per cell; original magnification ×40 and ×100). (D) TRAP-positive multinucleated OCs were quantified (P < .01) and assayed by MTT (E).