Figure 3

Osteoclasts are induced by prostate cancer CM in a concentration-dependent manner, exhibit characteristic actin structure, and resorb calcified matrices. A) RAW 264.7 cells were first primed with 50 ng/ml RANKL for 2 days, then cultured for additional 2 days untreated (negative control), or exposed to different dilutions of PC3 CM , and the average number of osteoclasts was assessed. Data are means ± SEM; n = 3-10 experiments; *P < 0.05, ***P < 0.001 indicate significance compared to negative control as assessed by ANOVA following by Tukey posttest. B) RANKL-primed osteoclast precursors supplemented for 2 days with 10% CM of PC3 (top) or LNCaP (bottom) were fixed, actin filaments were labeled using Alexa Fluor® 568 phalloidin (red), and nuclei were counterstained with DAPI (blue). Representative images of actin rings in osteoclasts formed after exposure to prostate cancer CM. C) RAW 264.7 cells were placed on calcium phosphate substrates, primed with RANKL for 2 days, and exposed to 10% PC3 (top) or LNCaP (bottom) CM for 2 days. Top: Representative image of active multinucleated osteoclast (red arrow) forming a resorption pit (red outline). Bottom: Representative image of resorbed areas after osteoclasts were removed.