Invadopodia formation assays were performed as described previously9 (link)10 (link). In brief, 786-O cells were transfected with siRNAs targeting Arf6 or EPB41L5. Twenty-four hours after transfection, cells were plated onto a culture dish coated with Alexa 594-labelled gelatin film, and cultured for 16 h. Cells were then stained with a membrane-permeable dye, calcein AM (Invitrogen). Immunostaining of fixed cells was performed as described previously9 (link)10 (link). HA-EPB41L5 and Arf6-HA were visualized using an anti-HA antibody coupled with an Alexa 488-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch). The number of cells degrading the gelatin film was counted using a confocal laser-scanning microscope (Model A1R, Nikon). Inhibition of extracellular proteolytic activity was performed using the following protease inhibitor cocktail22 (link): GM6001 (50 μM; sc-203979, Santa Cruz Biotechnology), E64 (250 μM; E3132, Sigma-Aldrich), pepstatin A (100 μM; P5318, Sigma-Aldrich), leupeptin (2 μM; L9783, Sigma-Aldrich) and aprotinin (2.2 μM; A4529, Sigma-Aldrich).
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