Figure 2.

The effect of RalBP1 depletion on PC3 growth and metastasis. (A) Western blots demonstrating decreased RalBP1 protein levels in PC3 cells after infection of lentiviral-based RalBP1-targeting shRNA constructs, shRalBP1-1 and shRalBP1-3. (B) i) In vitro growth assessment of cells in panel A. Cell numbers were quantitated with Alamar blue at each time point. ii) In vivo subcutaneous tumor growth of cells in panel A. Cells in serum-free RPMI medium were mixed with Matrigel and were subcutaneously injected into the mouse flank region in five mice per group with two sites per mouse. Tumor volume was measured every week with a caliper. Error bars represent SD. *P < .05 compared with sh-nontarget control at 5 weeks. (C) Xenogen evaluation of metastatic tumor growth of five mice injected with either nontarget shRNA or two different RalBP1-targeting shRNA PC3 cells. Xenogen images shown were taken at 9 weeks after injection. Graphs show quantitation of Xenogen signal at 3, 5, 7, and 9 weeks after injection. *P < .05 compared with sh-nontarget control at 9 weeks. (D) Images of representative mouse mandibles dissected from mouse on necropsy. Arrows indicate where tumors are located. (E) High-resolution CT images of mouse mandibles. Dissected mouse mandibles were imaged under a high-resolution CT scanner to demonstrate bone destruction by PC3 tumors. Arrows indicate where tumors are located. (F) Xenogen evaluation of orthotopic tumor growth of six mice injected with either nontarget shRNA or RalBP1-targeting shRNA PC3 cells in mouse prostate. **P < .01 compared with sh-nontarget control at 6 weeks. Xenogen images shown were taken at 6 weeks after injection. (G) Mouse prostate tumor size measured by a caliper at the time of necropsy at 6 weeks. **P < .01 compared with sh-nontarget control at 6 weeks from mice described in panel F.