JN-2 inhibits 4T1-induced bone destruction. Six-week-old female BALB/c (n=7) mice were intraperitoneally injected with DMSO or JN-2 (10 mg kg−1 of body weight) every other day before 4T1 injection. After 7 days, 4T1 cells (1 × 104 cells in 5 μl PBS) were injected into the femoral marrow space through the femoral condyle with a 30-gauge Hamilton syringe. Then, DMSO or JN-2 was intraperitoneally injected every other day for 21 days. (a) The femurs were investigated by histological sectioning with H&E staining (top) and μCT analysis (bottom and right; *P<0.05, **P<0.01). (b) The femurs were further analyzed for cortical bone volume (left) and trabecular number (right). *P<0.05, **P<0.01. Scale bar is 1 mm. (c) Serum PYD levels were measured by ELISA (*P<0.05, **P<0.01). (d) Histological sections of the femurs were immunostained with anti-P65 antibody (brown). T, tumor; M, marrow. Scale bar is 500 μm (left) and 100 μm (right). (e) Histological sections were stained for TRAP activity to detect osteoclast cells, and TRAP-positive cells were analyzed (*P<0.05). T, tumor; B, bone. Scale bar is 200 μm. (f) Total RNA were extracted from the femurs, and CXCL10 and RANKL mRNA levels were analyzed by real-time PCR (*P<0.05).