R37112

MVNs were fixed with 4% PFA (Thermo Scientific, 11490570) for 1 h at RT, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) for 10 min at RT and blocked with 10% Donkey Serum (PAN-Biotech, PANP30-0101). MVNs were then stained with primary antibodies diluted 1:100-1:200 in blocking buffer, overnight at 4 °C. We used anti-CD31 (Abcam, ab24590), anti-PDGFRβ (Abcam, ab32570), anti-S100b (Sigma, S2532), anti-desmin (ab15200), anti-GFAP (Invitrogen, MA5-12023), anti-CLDN5 (Invitrogen, 341600), anti-ZO-1 (Thermo Fisher, 339100), anti-VE-cadherin (Abcam, ab33168), anti-laminin (Abcam, ab7463) and anti-collagen type IV (Sigma, MAB1910) antibodies. Devices were then washed 5 times with 1× PBS for >5 min, and stained with corresponding secondary antibodies Alexa Fluor 488 donkey anti-rabbit (Invitrogen, A-21206) or Alexa Fluor 647 donkey anti-mouse (Invitrogen, A-31571) diluted 1:250 in blocking buffer, overnight at 4 °C. Devices were washed again 5 times with 1X PBS for >5 min, stained with lectin Ulex europaeus agglutinin I (UEA I) Rhodamine (Vector Laboratories, RL-1062-2) or phalloidin (Invitrogen, R37112) and DAPI (Invitrogen, R37606), and washed overnight at 4 °C.

All microscope imaging was performed with a Zeiss LSM 780 or Olympus SD-OSR. To assess the F-actin organization after certain treatments, cells were seeded on a glass-bottom culture dish (D11130H, Matsunami). After culture with the peptides, cells were fixed with 4% paraformaldehyde phosphate buffer solution (PFA, 30525-89-4, Wako) for 10 min at room temperature, washed, and permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 15 min. Cells were washed with DPBS twice and incubated with ActinRed (Rhodamine-conjugated phalloidin, R37112, Invitrogen) or ActinGreen (Alexa-Fluor-488-conjugated phalloidin, R37110, Invitrogen) for 15 min at room temperature. Cells were washed with DPBS before being imaged. Morphological characteristics, including cell spreading area and perimeter area ratio, were quantified by Image J.
For live-cell time-lapse imaging, transfected HuH-7 cells were placed inside a stage-top incubator (Tokai Hit) and were tracked with a 100x/1.35 Silicon UPlanSApo objective for more than 12 h. 5.5-µm stack images were acquired every 20 min, and imaging parameters were adjusted to minimize photobleaching and avoid cell death.