MLN3897 blocks fusion of OC precursor cells. (A) Adherent PBMCs cultured with RANKL and M-CSF (50 ng/mL) were exposed to MLN3897 (10 nM) for the indicated time points. After 3 weeks, TRAP⁺ multinucleated cells were stained and counted. Results are expressed as number of TRAP⁺ multinucleated cells per well: each value represents the mean (± SD) of OCs per well of at least 3 wells. All experiments were performed independently at least 3 times. (B) CCR1 and CD14 coexpression was analyzed by flow cytometry on human PBMCs cultured with or without RANKL and M-CSF (50 ng/mL) for 7 days. (C) PBMCs were cultured in the presence of RANKL and M-CSF with or without MLN3897 (10 nM). At the indicated time points, cells were fixed and stained with hematoxylin/eosin. Cells with more than 3 nuclei were enumerated; images were obtained with a light microscope (Leica Microsystems, Wetzlar, Germany) and acquired through IM50 software (Leica Microsystems Imaging Solutions, Cambridge, United Kingdom). (D) At day 7, PBMCs stimulated with RANKL and M-CSF (50 ng/mL) in the presence or absence of MLN3897 (10 nM) were harvested and lysed; proteins were immunoblotted with anti-pERK, ERK, and c-fos antibodies.