Effect of bortezomib on osteoblast progenitors and osteoblastic cells. Colony-forming units–fibroblast (CFU-Fs) and CFU-osteoblasts (OBs) were evaluated by alkaline phosphatase and alizarin red staining, respectively, in long-term human BM cell cultures incubated in the presence or absence of bortezomib at a concentration ranging from 2 nM to 5 nM (A). Human osteoblastic cells MG-63 or HOBITs (2 × 10⁶) were incubated in the presence or absence of bortezomib (2 nM to 5 nM) for 48 hours. The number of dead and apoptotic osteoblastic cells was evaluated by a colorimetric method. Osteoblastic cells treated with TRAIL or stimulating anti-CD95 Ab (FAS) have been used as positive control (CON = control) (B). mRNA expression of the osteoblast-related markers was evaluated by RT-PCR in human PreOBs treated with bortezomib or vehicle for 24 hours (C). PreOBs were treated with bortezomib or vehicle for 48 hours; thereafter, collagen I and Runx2/Cbfa1 protein expression was evaluated by Western blot analysis in cell lysates and osteocalcin (OC) levels detected in conditioned media by ELISA. Graphs represent the mean OCs ± SD of 3 repeated experiments measured twice (D). The activity of the transcription factor Runx2/Cbfa1 was evaluated by an ELISA-based method in nuclear lysates of PreOBs and HOBITs treated for 48 hours with bortezomib (2-5 nM). Graphs represent the mean Runx2/Cbfa1 activation ± SD using 10 μg protein in 3 repeated experiments measured in triplicate (E). Both dephospho and phospho β-catenin were determined by Western blot in cytosolic or nuclear lysates of PreOBs treated with bortezomib (2-5 nM) or vehicle. Actin and histone H1 were used as internal controls (F).