Figure 3
Mena11a downregulation affects migration, morphology and membrane protrusion.
(A,E): Western blot analysis of lysates from (A) T47D and (E) SKBr3 cells with stable, isoform-specific knockdown of Mena11a using two different shRNAs (sh-1, sh-2, respectively) and control shRNAs (sh-1C, sh-2C). Membranes were probed with anti pan-Mena and anti Mena11a antibodies. α-Tubulin was used as the loading control. (B,F): Quantitative analysis of the relative ratio of Mena11a: α-Tubulin as determined by densitometry in (B) T47D and (F) SKBr3 control and Mena11a-specific knockdown cells. Fold change in expression is relative to the appropriate control. Error bars: SEM, results represent triplicates. (C,D,I,J): Wound healing assay using T47D control (sh-1C, sh-2C) and Mena11a-specific knockdown (sh-1, sh-2) cells. (C) DIC images of cells after 0 and 48 hours in complete media. Scale bar, 50 μm. (D) Percent gap closure of cells after 48 hours in complete media. (G,H): Wound healing assay using SKBr3 control (sh-1C, sh-2C) and Mena11a-specific knockdown (sh-1, sh-2) cells. (K,L): MCF7 control and Mena11a-specific knockdown cells stimulated with 100 ng/ml Neuregulin-1. Quantitative results in (D,H,J,L) represent triplicates, error bars represent SEM. Unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.005. (G) DIC images of cells after 0 and 24 hours in complete media. Scale bar, 50 μm. (H) Percent gap closure of cells after 24 hours in complete media. (I) Morphology of cells. DIC images of the gap’s free edge after 24 hours. Scale bar, 50 μm. Insets are 9x magnification. (J) Morphometric analysis of cells. DIC images of the gap’s free edge after 24 hours. Circularity = 
Box and whisker plots of circularity; >470 cells analyzed. (K) Membrane protrusion kinetics; >22 cells analyzed. (L) Membrane protrusion of control and Mena11a-specific knockdown cells at t = 7 minutes post stimulation; >22 cells analyzed. Cells were starved for 4 hrs prior to stimulation with 100 ng/ml NRG-1.
