Figure 2.

Oprozomib and carfilzomib inhibit OC differentiation and function in vitro. (a) Human OCs were generated from PBMCs cultured in osteoclastogenic medium for 21 days in the presence or absence of indicated concentrations of the PIs (continuous treatment, left panel); alternatively, PIs were present only for the initial 4 h of differentiation (transient treatment, right panel). OCs were identified as multinucleated (≥3 nuclei) TRAP + cells. Representative micrographs of differentiated OCs after transient treatment are shown. (b) To assess inhibition of mineralized matrix resorption, PBMCs were seeded on calcium-coated slides and maintained in osteoclastogenic medium for 17 days with or without indicated concentrations of the drugs. Graphs represent mean values of samples from OCs derived from three healthy donors±s.d. *P<0.05 between treated cultures and vehicle control. Bar =50 μm. (c) PIs disrupted the integrity of the actin ring in multinucleated pre-OCs (14 days in osteoclastogenic medium under continuous PI-treatment). Actin =phalloidin–rhodamine, DAPI =nuclei; representative micrographs are reported. Bar =50 μm. (d) Pre-OCs were treated with indicated concentrations of PIs for 3 h prior to stimulation with RANKL for 30 min. In absence of PIs (vehicle), RANKL induces p65 translocation to the nucleus, whereas in PI-treated cells the p65 subunit of NF-κB is retained in the cytoplasm. p65 subunit =visualized in red, DAPI =nuclei; representative micrographs for each PI are shown (Bar =12.5 μm).