Figure 4. Wnt5A increases filamin A cleavage.

(a) The expression and localization of filamin A (red) was examined in melanoma cell lines of differing metastatic potential using immunofluorescent confocal microscopy. Filamin A is found in a regular pattern around the periphery of the cell in G361 cells (low metastatic potential), but is more cytoplasmic and irregular in the cell lines with a higher metastatic potential. (b) Treatment of G-361 cells with recombinant Wnt5A for 16 hours resulted in a redistribution of filamin A to the cytoplasm of the cells akin to the lines with higher metastatic capacity. Scale bar=20 μm. (c) Total protein lysates from G-361, UACC903, M93-047, and UACC647 cell lines were analyzed by western blot for the presence of filamin A cleavage fragments. Filamin A cleavage fragments were identified as 190 kDa (N-terminal) and 90 kDa (C-terminal) bands, and cleavage was indeed increased in cell lines with higher metastatic capacity. β-Tubulin was used as a loading control. (d) G-361 cells were treated with increasing amounts of recombinant Wnt5A for 16 hours and total protein was isolated for western blot analysis of the 190 and 90 kDa N-terminal cleavage fragment. β-Tubulin was used as a loading control.