Leukotriene B4 initiates osteoclastogenesis independent of receptor activator of nuclear factor κB ligand signaling. (a) and (b) Gene expression analysis of human peripheral blood mononuclear cell (PBMCs) cultured with macrophage colony-stimulating factor (M-CSF), or M-CSF + leukotriene B4 (LTB4), for 8 or 14 days, showing the differential expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1), cathepsin K, tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase 9 (MMP9) and β3 integrin. (b) Cytochemical staining for TRAP in human PBMCs cultured with M-CSF, M-CSF + RANKL, M-CSF + LTB4, and M-CSF + RANKL + LTB4 for 14 days, showing the number of TRAP+ multinuclear cells (three or more nuclei) per frame of view. (c) Imaging of TRAP cytochemical staining and phalloidin staining in PBMCs cultured with M-CSF, M-CSF + RANKL, or M-CSF + LTB4 for 14 days (scale bars represent 20 μm). Representative data and images from three experiments are shown. *P < 0.05 and **P < 0.01.