ZMEL cells form Paxillin-positive focal adhesion structures in vitro and in vivo. (A) Representative examples of positive fibronectin immunostaining of zebrafish larvae with transplanted ZMEL-GFP cells (left). Untransplanted larvae are similarly immunostained and used as a control to show the absence of fibronectin immunostaining in the same tissue (right). Scale bar is 10 µm. (B) Two TEM micrographs of untransplanted larvae at the skin region, 5 dpf, lateral view. Dashed white lines outline the skin ECM. Left panel indicates the absence of cells underneath the matrix and right panel indicates a pigmented melanocyte underneath the matrix. Scale bars are 1 µm. (C) Schematic of the process to generate primary ZMEL Paxillin-EGFP lines. A MiniCoopR plasmid expressing GFP-tagged zebrafish paxillin-a (pxna) is injected into single-cell stage embryos of Tg(mitfa:BRAF V600E); p53(lf); mitfa(lf). The melanocyte-rescued larvae are sorted and raised into adulthood for melanoma development (red arrowhead indicates melanoma tumor on adult zebrafish, middle panel). ZMEL Paxillin-EGFP cells are isolated from zebrafish melanoma tumors and cultured in cell culture dishes in vitro. Live imaging reveals Paxillin localizes to focal adhesions under in vitro cell culture conditions. Scale bar is 1 cm for melanoma-bearing zebrafish and 10 µm for the ZMEL Paxillin-EGFP cell. (D) Quantification of focal adhesion size in ZMEL Paxillin-EGFP cells in culture compared with in vivo. Non-parametric unpaired t test, Mean ± SD. n = 234 focal adhesions (12 cells) in vitro and n = 42 focal adhesions in vivo (4 cells).