Prostate cancer-derived factors induce activation of MEK/ERK pathway in osteoclast precursors. A) RANKL-primed RAW 264.7 cells were cultured for 5–60 min untreated (negative control), treated with RANKL, or exposed to 10% PC3 or LNCaP CM. Total protein was extracted and the levels of phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, and α-tubulin were assessed by immunoblotting. B) RANKL-primed RAW 264.7 cells were pretreated for 1 h with 0.1% DMSO vehicle, or 100 μM MEK inhibitor (PD98059), washed and cultured for 30–60 min untreated, treated with RANKL, or 10% PC3 or LNCaP CM. Total protein was extracted and immunoblotted for p-ERK1/2, total ERK1/2 and α-tubulin. C-E) RANKL-primed RAW 264.7 (C-D), or bone marrow (E) cells were pretreated for 1 h with 0.1%, DMSO vehicle (gray bars), or MEK inhibitor, 100 μM for RAW 264.7 and 50 μM for bone marrow cells (white bar), washed and cultured for 2 days (negative control), treated with RANKL, or 10% PC3 or LNCaP (right column), and the average osteoclast numbers were assessed. C) Representative images of osteoclasts formed from RAW 264.7 cells in different conditions. D-E) Average number of osteoclasts formed in RAW 264.7 (D) or bone marrow (E) cultures at different conditions. Data are means ± SEM; n = 4-10 experiments for RAW 264.7 cells, n = 3-7 experiments for bone marrow cells; *P < 0.05, ***P < 0.001 indicate significance compared to negative control; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to no inhibitor, assessed by Student’s t-test.