(a) Immunoblot analysis showing NUMB expression in metastatic melanoma cell lines with a control vector (shLuc) or shNUMB #1 and #2. Blotting for b-actin serves as a loading control. The relative intensities of the immunoblot bands were quantified. (b) Downregulation of NUMB by shRNA in metastatic melanoma cell lines leads to morphological changes, which were quantified. Bar = 200 mm. (c) shNUMB-transduced cells or control vector–transduced cells were grown as spheroids embedded in a 3D collagen matrix to test their invasive ability. NUMB knockdown cell lines showed increased invasion in WM1799 (on day 1) and WM3451 (on day 2) melanoma cell lines compared with that in the control cell lines. Bar = 200 mm. 3D, three-dimensional; pix, pixel; shLuc, short hairpin RNA targeting firefly luciferase; shNUMB, short hairpin RNA targeting NUMB; shRNA, short hairpin RNA.