a, Migration of A375 human melanoma cells stably expressing an shRNA directed against NECTIN1 (shNECTIN1) relative to cells expressing a control shRNA (shCTRL) in a transwell assay upon treatment with a FAK inhibitor (FAKi, PF562271) or a SRC inhibitor (SRCi, dasatinib). Cells were allowed to migrate for 6 h. Data represent mean ± s.d. of three independent experiments. Paired two-tailed t-test. b, Migration of six human melanoma cell lines stably expressing an shRNA directed against NECTIN1 (shNECTIN1) relative to cells expressing a control shRNA (shCTRL) in a transwell assay upon treatment with two independent FAK inhibitors (FAKi#1, PF562271; FAKi#2, GSK2256098). Cells were allowed to migrate for different times depending on the cell line (Extended Data Fig. 4i and Methods). Data represent mean ± s.d. of four independent experiments (paired two-tailed t-test). c, Western blot analysis of FAK and SRC phosphorylation levels in the cells described in panel a in the absence of serum. C, shCTRL; Sh, shNECTIN1. Representative example of five independent experiments. Signal intensity was normalized to GAPDH and presented as a ratio to the vehicle-treated shCTRL condition. Data represent mean ± s.d. (paired two-tailed t-test). d, Representative images of sections of adult casper recipients transplanted with control- or nectin1-knockout primary zebrafish melanomas, stained for phospho-fak by immunohistochemistry. Scale bar, 50 μm. e, Quantification of phospho-fak (p-fak) staining intensity in casper recipients transplanted with nectin1-wild-type (n = 19) or nectin1-knockout (n = 28) zebrafish melanomas. Data represent mean ± s.d. (two-tailed t-test). f, Number of 16-week-old zebrafish with disseminated melanoma cells in the indicated organs. g, Quantification of organ involvement in 16-week-old zebrafish bearing nectin1/ctrl-knockout (n = 9) or nectin1/fak-knockout (n = 9) melanoma. Data represent mean ± s.d. (two-tailed t-test). h, Representative images of adult casper zebrafish transplanted with nectin1-knockout primary zebrafish melanoma cells expressing a control gRNA (ctrl-knockout) or a gRNA targeting fak (fak-knockout). Insets show ×2 magnification views. Arrowheads indicate patches of disseminated melanoma cells. Scale bar, 1 cm. i, Quantification of the proportion of secondary recipients of nectin1/ctrl-knockout (ctrl-KO, n = 7) or nectin1/fak-knockout (fak-KO, n = 6) tumors showing tumor spreading. Data represent mean ± s.d. (two-tailed t-test).
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