Figure 2
EGF exposure, like LPS treatment, positively affects the cell surface localization of alpha-enolase supporting cell invasion. (a) HB2 and MCF-7 cells were treated with 0.1 µg/ml of EGF or 5 µg/ml of LPS for 24 hours, then double-stained with antibodies against alpha-enolase (α-Eno, green, live staining) and E-cadherin (red, staining after cell fixation), and visualized by confocal microscopy. The merged images (right panels) indicate co-localization of the two proteins. (b) Fluorescence signals were measured in control and treated cells using the ImageJ image-processing program. Each bar represents the mean ± standard deviation of three independent experiments. (c–f) Matrigel invasion assays. Unstimulated (Control) and EGF- or LPS- stimulated cells were allowed to invade for 48 hours, (c) representative images of cells on the underside of the transwell membrane and (d) quantification of cells that invaded. (e,f) Effect of anti-alpha-enolase antibodies on migration through Matrigel-coated transwell filters. MCF-7 cells were seeded in serum-free medium containing isotype-control antibody (isotype Ab) or anti-alpha-enolase monoclonal (α-Eno mAb, 50 µg/ml) and polyclonal (α-Eno pAb, 15 µg/ml) antibody and incubated in the absence or presence of EGF (0.1 µg/ml) or LPS (5 µg/ml) for 24 hours. (f) Quantification of invaded cells is shown relative to the untreated control, set at 1. Results are from three independent experiments, error bars represent standard deviation and p values (*P < 0.05, **P < 0.01, ***P < 0.001) indicate statistical significance.
