Figure 5.

RAD6B knockout decreases migration, invasion, and tumorigenic potentials. A and B: Migratory (A) and invasive (B) potentials. Assays were performed in triplicate, and results are expressed relative to control from two independent experiments. C: Immunofluorescence analysis of vimentin (Texas Red) and F-actin [fluorescein isothiocyanate (FITC)–phalloidin] filaments. Note the selective loss of vimentin from the lamellipodia of RAD6B knockout (KO) clones compared with controls. D: Colony formation. Colony-forming efficiency was expressed relative to control cells. Assays were performed in triplicate, and results are expressed from two independent experiments. E: Vertical scatter plots of excised tumor mass at time of sacrifice. Data analyzed by two-tailed t test (P = 0.0096). Note the characteristic melanoma pigmentation in control xenografts and its loss in RAD6B knockout xenografts. F: Hematoxylin and eosin analysis. Giant multinucleated cells characteristic of mitotic catastrophe (black arrow) and cells with aberrant mitotic shape (yellow arrow) in RAD6B KO clone are indicated. Mitotic (white arrow) and pigment-producing (red arrow) cells in control tumors are indicated. G: Pigmented metastatic foci (arrow) in lungs (top panel) and tumor cells derived from lung digests (bottom panel) of control M14 tumor-bearing mice. H: Quantification of metastatic foci on the surfaces of lungs. Results are expressed as means ± SD (A, B, D, and H). N = 3 (H). ∗∗∗P < 0.001. Original magnification, ×40 (F).