Figure 3.

Protrudin-KO cells display small and short invadopodia. (A) Cell lines as indicated in the figure were grown on coverslips; stained with anti-TKS5, anti-GFP, and phalloidin/Alexa Fluor 647 (actin); and analyzed by confocal microscopy. Note the undersized invadopodia in cells lacking Protrudin. (B) Cell lines as indicated in the figure were grown on coverslips, stained with anti-TKS5, anti-GFP and phalloidin/Alexa Fluor 647 (actin) and analyzed by confocal microscopy. Micrographs show orthographic and parallel maximum projections of z-stacks. In the parallel projections, the fluorescent intensity of TKS5 and actin were measured along a dotted line overlaying two invadopodia in each cell line, showing a reduced intensity of bothTKS5 and actin in cells lacking Protrudin as indicated in the intensity plots. (C) MDA-MB-231 and MDA-MB-231-Protrudin KO#2 cells were grown on coverslips coated with Oregon Green gelatin for 4 h, stained with anti-TKS5 and phalloidin/Alexa Fluor 647 (actin), and examined by superresolution microscopy (Airyscan). Z-stacks of individual cells were captured. Micrographs show section #9 from the parallel plane, and orthographic sections of the areas indicated by the dotted line. The graph shows the average length of TKS5-positive invadopodia per cell, quantified from Airyscan confocal z-stacks as described in Materials and methods. Each plotted point represents one cell, mean ± SD; parental, n = 19 cells; Protrudin KO, n = 15 cells from three independent experiments. ***, P < 0.0001, unpaired two-sided t test.