MLN3897 inhibits OC formation and function. Adherent PBMCs were cultured for 3 weeks in the presence of RANKL and M-CSF (50 ng/mL) with or without MLN3897 (0.2-10 nM). (A) Cells were stained for TRAP activity. Multinuclear TRAP⁺ cells are expressed as percentage of control. (B) Adherent PBMCs were cultured on dentine slices. After 3 weeks, resorption areas were stained with toluidine blue, and pit areas were quantified by light microscopy using the public domain NIH Image J program. Each value represents the mean (± SD) of resorptive areas of at least 3 wells, expressed as a percentage of total area. Images were obtained using a Leica DM IL microscope equipped with a 4×, 10×/0.22, and 20×/0.40 numeric aperture objective lens (Leica Microsystems, Wetzlar, Germany) and acquired through IM50 software (Leica Microsystems Imaging Solutions, Cambridge, United Kingdom). All experiments were performed independently at least 3 times. (C) After 3 weeks, we investigated cathepsin K expression on OCs obtained from 2 different donors and treated with MLN3897 (10 nM). Cells were harvested with cell dissociation buffer and lysed; proteins were subjected to immunoblotting with anti–cathepsin K antibody. To ensure equal protein loading, membrane was blotted for tubulin expression.