Figure 4
Mcl-1 levels decrease rapidly in mouse osteoclasts following cytokine starvation but are restored by pro-survival factors. Mouse osteoclasts were generated by culturing bone marrow macrophages for 5 days with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappa-B ligand (RANKL). (a) Osteoclasts were starved of M-CSF and RANKL for 8 hours and then fixed and processed for scanning EM analysis. Representative osteoclasts from non-starved (control) or starved cultures are shown. Bars = 10 μm. (b) Mouse osteoclasts were starved of M-CSF and RANKL for 2 to 12 hours. Western blot analysis was then used to determine the level of Mcl-1, cleaved caspase-3, Bcl-2, and Bcl-xL at each time point. (c) After osteoclasts were generated, the medium was replaced with normal medium (control [Ctrl]: M-CSF + RANKL), with medium lacking cytokines (starved), or with medium containing recombinant M-CSF, RANKL, tumour necrosis factor-alpha (TNF-α), or lipopolysaccharide (LPS). After 12 hours, Western blotting was used to determine the level of Mcl-1 in 40 μg of cell lysate. Data shown are representative of three independent experiments.