The P97 inhibitor CB-5083 suppressed osteosarcoma cell line proliferation. (A) P97 protein expression in 11 osteosarcoma cell lines (U2OS, U2R, ZOS, ZOSM, HOS, MNNG/HOS, 143B, SJSA-1, SAOS-2, MG63, and G292) and the human osteoblast cell line hFOB1.19. (B) Normalization of P97 protein expression by comparing the grayscale values of the Western blot bands. (C and D) Cell viability assay of 11 osteosarcoma cell lines treated with CB-5083. The cells were seeded in 96-well plates, cultured overnight and then treated with different concentrations of CB-5083 for 72 hours. The viable cells were detected using the MTT assay (C), and the IC50 values (D) were calculated. (E and F) Colony formation assay of SJSA-1 and U2OS cells treated with CB-5083. The cells were treated with CB-5083 for 24 hours, seeded at 500 cells/well in 6-well plates and cultured for 12 days. The cell colonies were the photographed, and the colonies that contained more than 50 cells were counted. *P<0.05, **P<0.01, and ***P<0.005 compared with the control group.