Figure 4.

C646 inhibits migration, invasion and the lung metastases induced by Rab22a-NeoF1 in vitro and in vivo. (A, B) ZOS-M cells were treated with 5 µM C646 (A) or 5 mM salicylate (B) for 24 h, cell lysates were subjected to IP using anti-K7ac-Rab22a-NeoF1 antibody, and then were analyzed by Western blotting by mAb RAD5-8. (C, D) Quantification analyses of migration and invasion assays using U2OS cells (C) or U2OS/MTX300 (D) cells stably expressing Vector, Rab22a-NeoF1 (WT) or its K7R mutant (K7R), as indicated, with or without the treatment of 5 µM C646 for 24 h. (E-H) The orthotopic osteosarcoma metastasis model in vivo using the U2OS/MTX300-Luc cells stably expressing Vector, Rab22a-NeoF1 (WT) or its K7R mutant (K7R), as indicated. After cell injection into mice for 3 weeks, the mice were intraperitoneally injected with C646 (10 mg/kg/d) or the control (7.7% DMSO+40% PEG300+ddH2O) daily for 14 days. Lung metastasis were detected by in vivo fluorescent imaging two months after cell injection. Representative images of mice (E). H&E staining of the lungs from representative tumor-bearing nude mice (F). Quantification analyses of Log₁₀(Average Radiance) of lung metastasis (G). Quantification analyses of lung nodules (H). Quantification analyses of wet lung weight (I) from the nude mice used in E.