Fig 1: rSj-Cys inhibits the expression of osteoclastogenesis-related genes and proteins. RAW264.7 cells were co-stimulated with M-CSF (25 ng/ml) and RANKL (30 ng/ml) and then treated with or without rSj-Cys (0.3 µM) for different time periods (24, 48 or 72 h). The expression levels of genes and proteins associated with osteoclast phenotype markers (CTSK, TRAP, ITGB3) and cell surface receptors of osteoclast precursors (RANK, OSCAR, TREM-2) were determined by (A) reverse transcription-quantitative PCR and (B) western blot analysis. Band intensity was semi-quantified with ImageJ software and normalized against β-actin levels. Data are presented as the mean ± SD of three independent experiments. ##P<0.01, ###P<0.001 vs. M-CSF alone group; *P<0.05, **P<0.01, ***P<0.001 vs. M-CSF + RANKL group. rSj-Cys, recombinant Schistosoma japonicum cystatins; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand; CTSK, cathepsin K; TRAP, tartrate resistant acid phosphatase; ITGB3, integrin β3; OSCAR, osteoclast-associated receptor; TREM-2, triggering receptor expressed on myeloid cells 2; NS, no significance.