Figure 4

Soluble factors produced by prostate cancer cells increase osteoclast formation in RANKL-independent manner. A, B) RAW 264.7 cells were primed with RANKL for 2 days, then cultured for 2 days untreated (negative control), with RANKL (positive control) or exposed to 10% PC3 or LNCaP CM, in the absence (black bars) or presence of 500 ng/ml OPG (white bars), and the average number of osteoclasts was assessed. A) Representative images of osteoclasts induced by RANKL or prostate cancer CM in the absence (top), or presence (bottom) of OPG. B) Average number of osteoclasts formed in different conditions. Data are means ± SEM; n = 4-10 experiments, except for RANKL and OPG, where n = 2 repeats. C-E) Bone marrow cells were primed with RANKL for 3 days, and cultured for 2 days untreated (negative control), treated with RANKL (positive control) (C) or exposed to 10% CM of PC3 (D) or LNCaP (E) cells, in the absence (black bars), or presence of 500 ng/ml OPG (white bars), or 5 μg/ml anti-MCSF blocking antibody (light gray bars) or TβRI inhibitor (5 μM, dark gray bars) and the average osteoclast numbers were assessed. OPG and anti-MCSF were added to prostate cancer CM for 30–60 min prior to addition to osteoclast precursors, TβRI inhibitor was added to the osteoclast precursor cultures for 60 min prior to addition of prostate cancer CM. Data are means ± SEM; n = 3-7 experiments. *P < 0.05, ***P < 0.001 indicate significance compared to negative control; ^#P < 0.05, ^###P < 0.005 indicate significance as compared to no inhibitor as assessed by Student’s t-test, no significant difference between samples treated with CM with and without OPG.