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. Author manuscript; available in PMC: 2011 Aug 4.

Published in final edited form as: Curr Biol. 2009 Nov 5;19(22):1875–1885. doi: 10.1016/j.cub.2009.09.059

(A) Parental LOX, LOX^ARF6-GTP and LOX^ARF6-GDP cells lines were fixed and stained with rhodamine phalloidin to visualize actin filament distribution or β1-integrin antibody, as indicated. Panels show the range of phenotypes in the LOX cell lines. Bar: 10µm. (B) Parental LOX, LOX^ARF6-GTP and LOX^ARF6-GDP cells in culture at low confluency were imaged using phase-contrast microscopy. Arrows point to shed vesicles in the growth media of LOX and LOX^ARF6-GTP cells. Bar: 10µm. (C) Vesicles released into the growth media from 2.2 × 10⁶ LOX, LOX^ARF6-GTP and LOX^ARF6-GDP cells, were isolated and probed for ARF6 and β1 integrin by western blotting (left) or protein was quantitated (right). Cell lysates were probed for ARF6 and α-tubulin by western blotting. Lower and higher molecular weight ARF6 bands in LOX^ARF6 cells correspond to endogenous and HA-tagged exogenous ARF6 respectively. (D) LOX^ARF6-GTP and LOX^ARF6-GDP cells were plated on FITC-gelatin-coated coverslips and allowed to invade. The cells were fixed and stained for cortactin (blue) and actin (red) (upper panel), or β1 integrin (red) (middle and lower panels). Arrows point to invadopodia and arrowheads indicate shed vesicles. Note arborization phenotype of LOX^ARF6-GTP cells when the underlying gelatin is relatively thick (middle panel). Bar: 10µm. (E) LOX^ARF6-GTP and LOX^ARF6-GDP cells lines were analyzed by electron microscopy. Image in top panel shows heterogeneous vesicular structures near the surface of LOX^ARF6-GTP cells. Lower panel shows vesicular structures that appear to stud the surface of LOX^ARF6-GDP cells. Bar: 1000nm. (F) Vesicles released into the growth media from an equivalent number (1.5 × 10⁶ cells) of indicated tumor cells lines in culture were isolated and probed for ARF6 by western blotting.

(A) Parental LOX, LOX^ARF6-GTP and LOX^ARF6-GDP cells lines were fixed and stained with rhodamine phalloidin to visualize actin filament distribution or β1-integrin antibody, as indicated. Panels show the range of phenotypes in the LOX cell lines. Bar: 10µm. (B) Parental LOX, LOX^ARF6-GTP and LOX^ARF6-GDP cells in culture at low confluency were imaged using phase-contrast microscopy. Arrows point to shed vesicles in the growth media of LOX and LOX^ARF6-GTP cells. Bar: 10µm. (C) Vesicles released into the growth media from 2.2 × 10⁶ LOX, LOX^ARF6-GTP and LOX^ARF6-GDP cells, were isolated and probed for ARF6 and β1 integrin by western blotting (left) or protein was quantitated (right). Cell lysates were probed for ARF6 and α-tubulin by western blotting. Lower and higher molecular weight ARF6 bands in LOX^ARF6 cells correspond to endogenous and HA-tagged exogenous ARF6 respectively. (D) LOX^ARF6-GTP and LOX^ARF6-GDP cells were plated on FITC-gelatin-coated coverslips and allowed to invade. The cells were fixed and stained for cortactin (blue) and actin (red) (upper panel), or β1 integrin (red) (middle and lower panels). Arrows point to invadopodia and arrowheads indicate shed vesicles. Note arborization phenotype of LOX^ARF6-GTP cells when the underlying gelatin is relatively thick (middle panel). Bar: 10µm. (E) LOX^ARF6-GTP and LOX^ARF6-GDP cells lines were analyzed by electron microscopy. Image in top panel shows heterogeneous vesicular structures near the surface of LOX^ARF6-GTP cells. Lower panel shows vesicular structures that appear to stud the surface of LOX^ARF6-GDP cells. Bar: 1000nm. (F) Vesicles released into the growth media from an equivalent number (1.5 × 10⁶ cells) of indicated tumor cells lines in culture were isolated and probed for ARF6 by western blotting.