Figure 1: WIP and WIRE are necessary for efficient degradation and migration in 3D matrices (circular invasion assays, CIA). | Scientific Reports

Figure 1: WIP and WIRE are necessary for efficient degradation and migration in 3D matrices (circular invasion assays, CIA).

From: WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion

Figure 1

(a) shControl, shWIP, shWIRE and double shWIP/WIRE knock-down MDA-MB-231 cells were cultured with Matrigel (25 μM GM6001 or carrier, DMSO). White lines in time-lapse microscopy images indicate the cell front at t = 0 and 18 h. (b) The cell-free area was measured and relative invasion was normalized to invaded area in control samples. (c) Time-lapse images of cell protrusions. Bars: 50 μm. (d) Quantification of speed, accumulated and Euclidean distances, and directionality of leading cells invading Matrigel. (e) Cells were incubated (24 h) before fixing in 4% paraformaldehyde and immunofluorescence (IF) analyses (cells stained for paxillin (green), cortactin (cyan) and F-actin (red)). Arrows indicate the direction of invading cells; arrowheads show invasive protrusions where paxillin, cortactin and F-actin localize. Bars: 10 μm. (f) Quantification of cell total corrected fluorescence (CTCF, top) and of the area occupied by paxillin-positive puncta compared to total paxillin (bottom) (see Methods). (g) IF images of cells invading Matrigel, stained for F-actin (red) and nuclei (TO-PRO, blue). Bars: 10 μm. (h) Quantification of length/width ratio of leading cells invading Matrigel. Length was calculated as distance from the most distal protruding tip of the cell to the opposite base of the nucleus. Width was measured as the widest cell distance across the nucleus. Each dot represents a single cell. (i) Stably infected shControl, shWIP or shWIRE MDA-MB-231 cells were allowed to invade Matrigel plugs in an inverted invasion assay. Bars: 250 μm. After 4 d invasion, cells were stained with live marker calcein-AM (4 μM, 1 h, 37 °C) and serial optical sections (10 μm intervals) were acquired. Magnified images from z = 40 μm sections are shown (bottom). Bars: 100 μm. (j) Cell invasion was quantified as cell-covered area >20 μm and then normalized to control values. Data show mean ± SD of three independent experiments. *p < 0.05, **p < 0.01; ***p < 0.001 by 2-way ANOVA and Bonferroni’s post-hoc test (b), by 1-way ANOVA and Tukey’s post-hoc test (d,h,j) or Student’s t-test (f).

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