Ma3100

For immunofluorescent microscopy, HUVECs were labeled with primary antibodies overnight, followed by incubation with a suitable fluorophore-conjugated secondary antibody for 1 h. Specifically, goat anti-mouse-CD31 antibody (MA3100, 1:200; Invitrogen), goat anti-rabbit-alpha smooth muscle actin (α-SMA) antibody (ab124964, 1:300; Abcam) were used to stain the makers of HUVECs and VSMCs, respectively. Immunofluorescent images were obtained using a confocal microscope (A1R HD25, Nikon, Tokyo, Japan) with NIS-Elements BR software (version 3.2. Nikon).

For live cell invasion assay, the cells were stained with cell tracker red (C7025, Invitrogen, Carlsbad, CA, USA) and cell tracker green (C34552, Invitrogen, Carlsbad, CA, USA) in 5 µg/mL and incubated for 20 min at 37 °C. For immunostaining, Zonula occludens-1 (ZO-1) (40-2200), cluster of differentiation 31 (CD31) (MA3100), Ras homolog family member A (RhoA) (MA1-011), Ras-related C3 botulinum toxin substrate 1 (Rac1) (701942), hypoxia-inducible factor 1-alpha (HIF-1α) (MA1-516), and matrix metallopeptidase 9 (MMP-9) (MA5-15886) were purchased from Invitrogen and used following the manufacturer protocols. For nuclei staining, 4′,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich, St. Louis, MO, USA) was used. The samples were rinsed with DPBS and fixed using 4% paraformaldehyde in DPBS for 10 min at room temperature (RT). After permeabilization using 0.1% Triton X-100 in DPBS for 15 min at RT, samples were blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) in DPBS for 1 h at RT. Then, the samples were incubated with primary antibodies overnight at 4 °C. After washing with 0.1% BSA, the samples were incubated with secondary antibodies for 2 h at RT and nuclei were stained with 300 nM DAPI. A confocal laser scanning microscope (CLSM) (LSM 510-META, Zeiss, Oberkochen, Germany) was used for imaging and analyses.