AMIGO2 regulates EC viability and angiogenesis. (A–J) HUVECs were pretransfected with scrambled siRNA and AMIGO2-specific siRNA and harvested for analysis 48 h after transfection. (A) FAK immunostaining was performed in parallel. Images on the right are enlargements of the boxed regions on the left. Bars, 20 µm. (B) Cell viability of AMIGO2 siRNA–transfected HUVECs in complete media and under conditions of serum-free starvation with or without VEGF (n = 6). Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. Data are means ± SD. (C) Expression of active caspase-3 and FAK fragments in AMIGO2 siRNA–transfected ECs. (D) Different stages of apoptosis were detected by phycoerythrin-conjugated annexin V flow cytometry and PerCP–7-AAD staining. Quadrant gates are based on unstained, annexin V, or 7-AAD alone. Quadrant regions show the percentage of living cells (7-AAD⁻/annexin V⁻), early apoptotic cells (7-AAD⁻/annexin V⁺), late apoptotic cells (7-AAD⁺/annexin V⁺), and necrotic cells (7-AAD⁺/annexin V⁻). The data are representative of three experiments conducted by using different samples. (E and F) Wound healing migration and gelatin-coated Transwell migrations with or without VEGF were performed. Two types of siA2 were evaluated for wound healing migration. n = 3 and n = 6. (G) Matrigel-induced tube formation by AMIGO2 siRNA–transfected HUVECs was assessed. (H and J) Mean numbers of tubule branch points and of tubes per field in randomly selected images were quantified. (I) Tube length is presented as the percentage of total tube length per field versus that of untreated control cells. (H–J) Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. Data are means ± SD. n = 4. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. sc, scrambled siRNA; siA2, AMIGO2 siRNA.