CEP-18770 inhibits EC survival, proliferation and tubular morphogenesis and M-CSF-RANKL–mediated osteoclastogenesis. (A-C) HMEC and TEC cells were treated with CEP-18770 and bortezomib (BOR) at the indicated concentrations. (A) Apoptosis was measured at 24 hours by flow cytometry after staining with TMRM. (B) Proliferation was assayed at 48 hours by crystal violet staining. Absorbance was read at 595 nm with an ELISA reader. Error bars represent SD. (C) Representative micrographs of capillary-like structure formation on Matrigel-coated wells in the presence or absence of proteasome inhibitors. Cells were observed with a Nikon inverted microscope and experimental results recorded after 6 hours from seeding. (D) Data show the mean (± 1 SD) of total length of capillary-like structures analyzed by the Micro-Image system (Casti Imaging) and expressed as mm/field by the computer analysis system in 5 different files at 100× magnification in duplicated wells of 4 different experiments. (E,F) To obtain fully differentiated osteoclasts (OC), PBMC were cultured for 10 days in the presence of recombinant human M-CSF. OC were activated by RANKL and simultaneously treated with CEP-18770 (CEP, 20 nM) or BOR (10 nM) for 5 days. Cells were incubated in duplicate for each condition. Mature OC were identified as TRAP⁺ multinucleated cells containing 3 or more nuclei. (E) Histograms represent means (± SD; error bars) of absolute numbers of OC from 6 nonmyelomatous donors. **P < .01 as analyzed by Student t test. (F) Three representative photographs of OC treated with control diluent (Control), CEP-18770 20 nM (CEP), and bortezomib (BOR) 10 nM. Arrows indicate TRAP⁺ cells (final magnification 400×).