Figure 5. Mena11a expression dampens membrane protrusion but EGFR activation is unaffected.

(A–C): MTLn3 cells stably expressing GFP, Mena and Mena11a, stimulated with 5 nM EGF. (A) DIC images of membrane protrusion during stimulation. Red arrowheads: lamellipodial protrusions. Protrusions are evident in Mena cells, but dampened in Mena11a cells. (B) Membrane protrusion kinetics of cells after EGF stimulation. Error bars: SEM. (C) Membrane protrusion after t = 180 seconds. Error bars: SEM. Results represent triplicates, >90 cells analyzed. One-way ANOVA **p < 0.01, ***p < 0.005. (D) Kymographs from time-lapse movies of MTLn3 GFP and MTLn3 GFP-Mena11a cells stimulated for 300 seconds. Kymographs demonstrate lamellipodial activity; ascending contours of edge represent protrusions, while descending ones represent withdrawals. (E) Box and whisker plot quantifying the time of individual protrusions (left) and protrusion persistence (right) during stimulation. Data for MTLn3 GFP cells are from 112 protrusion events, for MTLn3 GFP-Mena11a cells from 90 protrusion events. Unpaired t-test ***p < 0.005. (F,G) (Left panel): Western blot analysis of MTLn3 cells stably expressing GFP, Mena and Mena11a. Cells were starved for 4 hours, then stimulated for 0, 0.5, 1, 2, 3, and 5 minutes with 5 nM EGF. Membranes were probed with (F) anti-EGFR pY1068 and (G) anti-EGFR pY1173. GAPDH used as loading control. (Right panel): Densitometry of the relative ratio of (F) EGFR pY1068/GAPDH and (G) EGFR pY1173/GAPDH as determined by densitometry. Fold increase is over baseline (no EGF stimulation).