Methotrexate suppresses osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) staining showing reduction in the number of TRAP-positive cells when CD14+ osteoclast precursors were incubated with supernatants from methotrexate (MTX)-treated osteoblast-like cells (A) as compared with supernatants from nontreated osteoblast-like cells (B); arrow indicates a TRAP-positive pre-osteoclast. The fold decrease in the number of TRAP-positive pre-osteoclasts in MTX-treated osteoblast-like cells as compared with supernatants from nontreated osteoblast-like cells (C). TRAP staining showed a reduction in the number of TRAP-positive cells when CD14+ osteoclast precursors were incubated with MTX (D) as compared with control (E) in the presence of exogenous macrophage colony-stimulating factor M-CSF and RANKL; arrow indicates a TRAP-positive multinucleated osteoclast. The fold decrease in the number of TRAP-positive pre-osteoclasts and TRAP-positive multinucleated (more than two nuclei) osteoclasts in MTX-treated cultures as compared with control cultures (F). Von Kossa brown stained osteologic discs (G) showing large clear resorption areas (arrow) that are reduced following MTX exposure (H). Inverted images with the black surface representing the bone resorption areas in the controls (I) and MTX-exposed cultures (J), allowing quantification of data as seen in the adjacent graph (K). *p<0.05.