FIGURE 4.

Sema7A upregulates endothelial cell migration and tube formation through FAK/MAPK signaling pathway. (A) Sema7A-pCDH-HUVECs and pCDH-HUVECs were treated with FAK inhibitor (PF573228, 10 μM) or MAPK inhibitor (U0126, 20 μM) for 24 h and then subjected to wound healing assay at selected time points. Phase contrast images are from the start of the assay (0 h) and after 24 h. Location of initial scratch margins indicated by dashed green lines. Bar = 100 μm. (B) Quantified migration distances from A for 24 h migration ability. Data are shown as mean ± SEM. Results are representative of ≥ 3 independent experiments. ^∗∗∗∗P < 0.0001. (C) Representative images of transwell membranes with cells stained by crystal violet. Cells were prepared as in (A). Bar = 50 μm. (D) Quantified numbers of migrated cells in Sema7A-pCDH-HUVECs and control pCDH-HUVECs. Data are shown as mean ± SEM. Results are representative of ≥ 3 independent experiments. ^∗∗∗∗P < 0.0001. (E) Sema7A-pCDH-HUVECs and control pCDH-HUVECs subjected to tube formation assay after pretreatment same as in (A). Bar = 50 μm. (F) Quantified data from (E) for tube formation numbers. Data are shown as mean ± SEM. Results are representative of ≥ 3 independent experiments. ^∗∗∗∗P < 0.0001. (G–I) P-FAK (G,H), P-ERK1/2 proteins (G,I) were analyzed by western blotting normalized to total FAK/ERK1/2 and displayed as fold-changes relative to control pCDH-HUVECs. Sema7A-pCDH-HUVECs and control pCDH-HUVECs were pretreated same as in (A). Data are shown as mean ± SEM. Results are representative of ≥ 3 independent experiments. A.U = arbitrary unit. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.