Evans blue e2129

The following drugs and antibodies were applied: metformin (M0605000), LY294002 (L9908), Evans blue (E2129), DAPI (D8417) and Alexa Fluor 488 goat-anti-rabbit secondary antiserum (SAB4600234) obtained from Sigma (USA). Monoclonal anti-NeuN primary antiserum was obtained from Merck Millipore (Darmstadt, Germany). Chromo 546-conjugated anti mouse-IgG, horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG were purchased from Abcam (Cambridge, MA, USA). Akt, phospho-Akt and β-actin were obtained from Chemicon.

LPS (from Escherichia coli) (L-2630) and FITC-dextran (53379) were purchased from Sigma-Aldrich. Endothelial cell growth medium (ECM, 1001) was purchased from ScienCell. Short hairpin RNA against autophagy-related gene 7 (shATG7) plasmids, ATG7 shRNA lentivirus particles and antibodies targeting ATG7 were purchased from Santa Cruz Biotechnology (sc-41448-SH, sc-41448-V, and sc-376212). The following antibodies, including anti-microtubule-associated protein 1-light chain 3 (LC3) B (3868), anti-sequestosome 1/p62 protein (p62) (5114), anti-GAPDH (2118) and anti-rabbit IgG (7074), were purchased from Cell Signaling Technology. mRFP-GFP-LC3 adenoviral vectors were obtained from Han Bio Technology Co. Ltd. (HanBio, Shanghai, China). Anti-zonula occludens-1 (ZO-1) and anti-claudin-5 were purchased from Abcam (ab216880 and ab131259). Rhodamine-conjugated phalloidin was purchased from Invitrogen (Molecular Probe, R415). The following enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems: tumor necrosis factor (TNF)-α (MTA00B), interleukin (IL)-1β (MLB00C), IL-10 (M1000B), vascular endothelial growth factor (VEGF) (MMV00), fibroblast growth factor (FGF) (MFB00) and epidermal growth factor (EGF) (MEG00). Evans blue (E2129) was purchased from Sigma-Aldrich.

MIO-M1, HUVECs, and ARPE19 cells were cultured in DMEM with 10% (vol/vol) FBS (Quality Biological) and 1% penicillin/streptomycin (Cellgro). A hypoxic chamber (Coy Laboratory Product Inc.; Two Glove Model) with 5% CO₂, 94% N₂, and 1% O₂ gas concentration at 37°C was used to culture hypoxic cells. The control cells were cultured in an incubator maintained at 37°C, with 21% O₂ and 5% CO₂. Digoxin was purchased from Tocris (4583/50). Acriflavine was obtained from MilliporeSigma (8048-52-0). DMSO (472301-500ML) and Evans blue (E2129) were purchased from Sigma-Aldrich, and 4-(6-bromo-1H-indol-3-yl)-2-(7-bromo-1H-indol-3-yl)thiazole, designated 32-134D, was synthesized as previously described (44 (link)). Aflibercept was obtained from the Johns Hopkins University Pharmacy.