Figure 5.
Effect of IFITM1 silencing on the metastatic formation of DMS273-GFP cells in nude mice. (A) Quantitative analysis of IFITM1 mRNA in silenced cells. One microgram of total RNA extracted from the cells was subjected to real-time RT-PCR. Data are expressed as the mean + SD of a triplicate experiment. (B) Western blotting for IFITM1. The cells were treated with 10 ng/mL interferon β (IFNβ) for 24 h and then lysed. Whole-cell lysates (20 µg) were separated by 15% SDS-PAGE, and membranes were blotted with anti-IFITM1 (top panel) and anti-α-tubulin antibodies (bottom panel, loading control). Relative IFITM1 expression was calculated from the signal intensity and normalized to α-tubulin levels. Silencing efficiency was calculated as the percentage of the relative expression level in shIFITM1 cells to that in shLacZ cells. (C) The in vitro growth rate of the silenced cells was determined using the MTT assay. The growth rate was calculated as the ratio of the absorbance of cultured cells to that of cells on day 0. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. (D–F) Orthotopic tumor growth and metastatic formation in mice with orthotopic tumors generated using DMS273-GFP control shRNA (shLacZ) (n = 10) or IFITM1-silenced (shIFITM1) cells (n = 9). In total, 2.5 × 105 cells were transplanted into the left lung of each nude mouse. Mice were sacrificed when they became moribund, and orthotopic and metastatic tumors were assessed. (D) Representative fluorescence images of the orthotopic tumor and bone metastases of mice transplanted with control shRNA (shLacZ) cells. Bar, 2 mm. (E) Distant metastatic tumor formation. The dotted lines represent the means of the number of metastasis-positive organs per mouse. Percentages show the incidence of distant metastasis in each group. * p < 0.05, Mann–Whitney U-test. (F) Orthotopic tumor formation. Results are expressed as the mean + SD. (G–H) Metastatic colony formation in the lungs of experimental metastasis models generated using DMS273-GFP control shRNA (shLacZ) (n = 22) or IFITM1-silenced (shIFITM1) cells (n = 20). In total, 1 × 106 cells were injected into the tail vein of each nude mouse. At 7–8 weeks post-inoculation, lungs were dissected, and metastatic foci were counted. (G) Representative fluorescence images of the lung metastases of both groups. Bar, 2 mm. (H) Percentages show the incidence of distant metastasis. * p < 0.05, Mann–Whitney U-test. # p < 0.05, Fisher’s exact test.