Fig. 2.

hMENAΔv6 localizes at focal adhesions and its overexpression increases the β1 integrin activation, whereas hMENA^11a transfection in hMENAΔv6-expressing BT549 cells induces morphological changes, inhibits β1 integrin activation, and the downstream signaling pathway. a Confocal analysis with the indicated antibodies (red) of BT549 cells transfected with hMENAΔv6-GFP (green). Magnification ×63. Scale bar = 20 µm. b Percentage of active cell surface β1 integrin (9EG7) with respect to total β1 integrin (TS2-16) analyzed by flow cytometry in DAL, BT549, and A549 cells transfected with empty vector (CNTR) or hMENAΔv6 showing that hMENAΔv6 transfection increases the activation of β1 integrin. Data are reported as the mean ± SEM of three independent experiments. p value was calculated by 2-tailed Student’s t test. The upper panel shows Western blot analysis with Pan-hMENA Ab. c Matrigel invasion assay performed on A549 cells (50,000 cells; 24 h of invasion), transiently transfected with the empty vector (CNTR) or hMENAΔv6, and treated with β1 integrin blocking antibody AIIB2 or with IgG1 isotype control toward serum. The assay was repeated three times, performed in triplicate each time. Standard deviations are indicated. p value was calculated by 2-tailed Student’s t test. Inset: WB analysis of the A549 cells to verify hMENAΔv6 transfection. d Phase-contrast microscopy images of BT549 cells transfected with empty vector (CNTR) and hMENA^11a-expressing cell clones (#14 and #104) when grown for 10 days in 3D on lrECM. Magnification ×40. e Percentage of active β1 integrin (9EG7) with respect to total β1 integrin (TS2-16) analyzed by flow cytometry in BT549 CNTR (hMENA^11a negative/hMENAΔv6 positive) or hMENA^11a-expressing clones, showing that hMENA^11a transfection reduces the activation of β1 integrin. Data are reported as the mean ± SD of three independent experiments. p value was calculated by 2-tailed Student’s t test. Representative histograms of flow cytometric staining for 9EG7 in BT549 CNTR or hMENA^11a--expressing clones are shown. f WB analysis of BT549 CNTR or hMENA^11a-expressing clones with the indicated antibodies, showing that hMENA^11a transfection reduces the phosphorylation of β1 integrin signaling partners, TALIN, SRC, and FAK. Immunoreactivity was determined by ImageJ and phosphoproteins were normalized in comparison with the respective total proteins (TALIN, SRC, and FAK). Numbers indicate the fold changes. The fold reduction of phoshorylated protein expression in hMENA^11a-expressing clones is reported on the right. Data are reported as the mean ± SD of three independent experiments