Figure 2.
Reduction in the expression level of βig-h3 in SW620 colon cancer cells inhibits metastasis in vivo. (A) The expression level of βig-h3 in SW620 cells was reduced by 78% and 69%, respectively, using two different shRNA constructs. SW620 cells (parental) were infected with recombinant retroviruses containing vector control (VEC), shRNA1 (βig-h3-shRNA1), or shRNA2 (βig-h3-shRNA2). An equal amount of concentrated supernatant from each cell type as indicated was examined for the presence of βig-h3 by Western blot using an anti-βig-h3 antibody. The whole-cell lysate from each sample of cells from which the supernatant was collected for the determination of βig-h3 expression was blotted with anti-tubulin antibody for loading control. (B) Reduced βig-h3 expression led to decreases in tumor formation on the back of mice by the SW620 cells. We injected 2 × 106 SW620-VEC, SW620-βig-h3-shRNA1, or SW620-βig-h3-shRNA2 cells through the tail vein into 5-wk-old SCID-Beige mice with five animals for each group. Mice were sacrificed and examined for the growth of metastatic tumors 7 wk after injection. Results are the mean ± SD of five animals from each group. Four independent experiments were performed. (*) P < 0.01 (n = 5). (C) Representative results from two independent experiments showing total numbers of lung metastatic nodules in individual mouse injected with SW620-VEC, SW620-βig-h3-shRNA1, or SW620-βig-h3-shRNA2 cells. The counting was performed under the dissection scope. (*) P < 0.01 (n = 10). (D) Representative results showing that injection of SW620-VEC cells or SW620-βig-h3-shRNA1 and SW620-βig-h3-shRNA2 via tail veins led to different patterns in the formation of metastatic tumors.