Fig. 2

RANKL-induced caspase activity promotes fusion of osteoclast precursors. a Representative confocal laser scanning microscopy images of bone marrow-derived osteoclasts differentiated for 72 h in medium containing 10% L929-conditioned supernatant and 50 ng/mL RANKL either in the presence or absence of 2 μmol/L staurosporine (STS). Cells were fixed and stained for phalloidin (blue), DAPI (white), cleaved caspase-8 (“Cl-casp 8”, red) and TUNEL (green). Scale bar: 30 µm. b Western blot analysis of total cell extracts obtained from bone marrow-derived osteoclasts differentiated for 72 h in medium containing 10% L929-conditioned supernatant and 0, 10, or 50 ng/mL RANKL. Antibodies against the full form (“Procaspase”) or the cleaved active form (“Active caspase”) of caspases 3, 8, and 12 were used. β-actin signal served as loading control. c Quantification of band intensity ratios between cleaved active and full form of the Western Blot shown in b. d, e Bone marrow-derived osteoclasts were differentiated for 72 h in medium containing 10% L929-conditioned supernatant and 10 ng/mL RANKL in the presence of 10 μmol/L caspase-3 inhibitor, 10 μmol/L caspase-8 inhibitor, or vehicle only. Cells were fixed and stained for TRAP. d Representative micrographs of osteoclast size and nuclei per cell. Scale bar: 200 µm. e Quantification of the fusion index defined as the number of nuclei per cell. For every treatment group of the experiment, the cumulative frequency in percent of each occurring fusion index was calculated (top). The average fusion index was then determined, normalized to vehicle-only control and quantified (bottom). Indicated is mean ± SEM. Unpaired, two-tailed Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001, n = 3 for each caspase inhibitor-treated group and n = 9 for control group. f Bone marrow-derived osteoclasts were seeded onto bone-coated osteoplates and differentiated for 72 h in medium containing 10% L929-conditioned supernatant and 10 ng/mL RANKL in the presence of 10 μmol/L caspase-8 inhibitor or vehicle only. Left panel: Representative micrographs showing resorbed area (black) vs. remaining bone (white). Scale bar: 200 µm. Right: Quantification of resorbed area vs. remaining bone area in percent. Indicated is the mean. Unpaired, two-tailed Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001, n = 4 per group. All images and data are representative of three independent experiments