Figure 5

Prostate cancer-derived factors induce calcium/NFATc1 signaling in osteoclast precursors. A, B) RANKL-primed RAW 264.7 cells were loaded with fura-2-AM, cultured for 15 min untreated (negative control), with 50 ng/ml RANKL, or 10% PC3 or LNCaP CM, and imaged for 2 min. A) Average basal calcium levels. Data are means ± SD; n = 32-48 cells per condition; *significance compared to negative control as assessed by Student’s t-test. B) Percentage of active cells classified as having standard deviation of basal calcium above 0.05. C) RANKL-primed bone marrow precursors were pre-treated with 50 μM BAPTA (white bars) and exposed to 10% PC3 or LNCaP CM for 2 days, and average number of osteoclasts was assessed. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control; ^#significance compared to no inhibitor. D, F) Protein was extracted or samples were fixed from untreated or RANKL-primed RAW 264.7 cells, or RANKL-primed precursors cultured for 2 h untreated, with RANKL, or with 10% PC3 or LNCaP CM. D) NFATc1 protein levels, α-tubulin as a loading control. E) NFATc1 localization (green); nuclei were counterstained using DAPI (blue). F) Average NFATc1 nuclear localization normalized to the continuous RANKL (RL) treatment. Data are means ± SEM; n = 3 experiments. G, H) RANKL-primed bone marrow cells were pretreated with 0.1% DMSO vehicle (grey bars), or 50 μM NFAT inhibitor VIVIT (white bars), and cultured untreated, treated with RANKL, or with 10% PC3 or LNCaP CM for 2 h to examine NFATc1 localization; or 2 days to assess osteoclast formation. G) Average percentage of cells with nuclear NFATc1. Data are means ± SEM; n = 3 experiments. H) Average number of osteoclasts. Data are means ± SEM; n = 3-7 experiments; *significance compared to negative control, ^#significance as compared to no inhibitor.