AZD6244 OC formation and transcripts of OC-stimulating factors in MM cells. (A) Adherent monocytic OC precursors from MM-patient PBMCs were incubated with M-CSF/RANKL, in the presence or absence of AZD6244 (0.1 μM), for indicated time intervals. Total cell lysates were prepared and subjected to immunoblotting using anti-pERK Ab, with anti-ERK Ab as a loading control. (B) Adherent OC precursors from MM patient PBMCs were incubated with M-CSF/RANKL in the presence of AZD6244 for 10 days. Total RNA was then prepared and subjected to RT-PCR for cathepsin K (PCR product: 127 bp). RT-PCR for GADPH served as an internal control. (C) PBMCs from MM patients (n = 3) were incubated with M-CSF/RANKL for 14 days, in the presence or absence of AZD6244. OC formation was characterized by integrin α_vβ₃ expression by flow-cytometric analysis. *P < .05; **P < .005; data represent the mean of 3 experiments (± SE). Multinucleated OCs were induced by M-CSF/RANKL (control), whereas AZD6244 (0.2 μM) blocked OC formation; original magnification × 100. See “Patients, materials, and methods; Immunohistochemistry” for more information on image acquisition. (D) Transcriptional changes of indicated OC-activating factors in MM1S cells following AZD6244 treatment.