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Fig. 3 Comparable WASF3-induced metastasis of DU145 cells in mice and fish. Knockdown of WASF3 in DU145 cells using two different shRNA constructs (shW3-1 and shW3-2) shows significant reduction in protein levels (a). DU145 cells transfected with a control shRNA (shGFP) show high level invasion in vitro(b) but knockdown of WASF3 in these cells using two different shRNAs (shW3-1 and shW3-2) leads to significantly reduced invasion. In fish (c), no, or reduced numbers, of WASF3 knockdown cells (shW3-1 and shW3-2) spread throughout the body of the fish, whereas the control cells (shGFP) spread extensively (arrows) (shown as z-stack images using 40 × magnification). Quantitation of invasion potential shows a significant reduction (** p >0.001) in the WASF3 knockdown cells compared with control shGFP cells (d, above). This reduced invasion is mirrored by the metastasis efficiency in cohort studies of fish (d, below). In mice (e), tumor nodules (arrows) on the surface of the lungs can be seen in the control shRNA treated cells compared with cells expressing shW3-1 (left) 3 months after tail veil injection. Histological examination of the lungs of these mice shows large tumor foci from the control cells (arrows) compared with small tumor foci for the WASF3 knockdown cells.