Fig. 2

Rictor supports metastasis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. a-c MCF10A, MCF10A-HER2, MCF10A.Rictor^ZFN and MCF10A. Rictor^ZFN-HER2 cells were assessed by western analysis a and for invasion through Matrigel-coated transwell filters (b, c). Cells were stained with crystal violet 24 h after plating in the upper well of transwell chambers, and imaged (representative images shown in (b). The numbers of cells on the bottom side of each transwell were counted in digital images by two independent investigators blinded to sample identity, and used to calculate the average number of cells invading to the lower side of each filter (c). Each data point represents the average value of three experimental replicates; midline represents the average of three biological repicates, error bars are the standard error. Student’s t test was used to assess significance. d-f SKBR3 and MDA-MB-361 cells stably expressing shRictor or shScr were assessed by western analysis (d) and for invasion through Matrigel-coated transwells (e, f), and quantitated as described in (b). g-h MDA-MB-361 and SKBR3 cells (5 × 10⁶) expressing scrambled short hairpin shRNA sequences (shScr) or short hairpin RNA sequences against Rictor (ShRictor) were delivered by tail vein injection to athymic Balb/C nu/nu female mice. Lungs harvested at 8 weeks post injection were assessed by histological analysis and imaged at × 40 (g); representative images shown, arrows indicate metastatic nodules). Mice were scored as positive or negative for metastatic nodules in the lung to determine the percentage of mice harboring metastatic nodules (h)