LOXL2 is functionally involved in the hypoxia-induced network assembly of collagen IV. (A-C) HUVECs transfected with control (shctl), lox (shlox), or loxl2 (shloxl2) shRNA were cultured in hypoxia. (A) Immunofluorescence detection of LOXL2 and collagen IV (Col IV) was performed. Nuclei were stained with DAPI (blue). (B) Semiquantitative RT-PCR of col4a1 and actin from HUVEC transfected with control, lox or loxl2 shRNA. (C) Overnight media were collected and subjected to immunoblotting for collagen IV (Col IV). (D-E) Loxl2-depleted HUVEC (shloxl2) were infected with control (ctl) or LOXL2 coding lentivirus (LOXL2). (D) Cell lysates and secretion media were subjected to immunoblotting for LOXL2 detection (IB LOXL2). (E) Immunofluorescence of LOXL2 and collagen IV (Col IV) in the ECM of EC incubated in hypoxia. Collagen IV assembly was analyzed by confocal microscopy (left panel) and quantified using ImageJ (right panel). (F-G) HUVEC were cultured in hypoxia in absence (ctl) or presence of β-APN for 5 days. (F) Duplicate ECM extracts were subjected to immunoblotting of collagen IV. (G) Immunofluorescence detection of LOXL2 and collagen IV (Col IV). (H-J) HUVECs transfected with control (shctl) or col4a1 (shcol4a1) shRNA. (H) Semiquantitative RT-PCR of loxl2, col4a1, and actin. (I) Collagen IV (Col IV) expression in HUVECs was detected by immunoblot in total lysate (cell and extracellular matrix). (J) HUVECs were coated on Cytodex beads, embedded in fibrin gels and maintained in the presence of fibroblasts. Images were acquired at day 6 for sprout length measurement. For each experiment (n = 3), total length of sprouts per bead was normalized to the mean of control cells. Graph represents the mean of normalized values ± SEM. ***P < .001.