Effect of bortezomib on osteogenic differentiation of human mesenchymal cells. Confluent PreOB cells were treated with or without bortezomib (2 nM) or BMP-2 (50 ng/mL) or PTH (1-34) (50 ng/mL) or MG-132 (100 nM) or MG-262 (10 nM). After 24 hours, collagen I, Runx2/Cbfa1, and GAPDH mRNA expression was evaluated by RT-PCR (A). Adherent PreOB cells were incubated in osteogenic differentiating medium as described in “Patients, materials, and methods” in the presence or absence of bortezomib (2 nM) or BMP-2 (50 ng/mL) or PTH (1-34) (50 ng/mL) or MG-132 (100 nM) or MG-262 (10 nM) for 21 days, replacing medium with fresh one every 3 days. After the culture period, cells were fixed and stained for the presence of bone nodules by alizarin red. Graphs and bars represent the mean plus SD of bone nodules × field of 2 independent experiments performed in triplicate (*P < .05; **P < .01) (B). Images were obtained on a Nikon Eclipse TE 300 microscope at 10×/0.13 using a DS-U1 digital slight and at 4×/0.12 objective lens (Original magnification × 5).