The human brain microvascular endothelial cell line was cultured as described in the previous studies (Chi et al., 2012). The human brain microvascular endothelial cell line was cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum, two mM glutamine, one mM sodium pyruvate, streptomycin (100 μg/mL), and penicillin G (50 μg/mL) at 37°C in 5% CO2.
Proteins were extracted from HBMEC, lysed using RIPA, which contains protease and phosphatase inhibitors, followed by centrifugation at 13,000 rpm for 15 min at 4°C. SDS-PAGE separated the samples. After blocking with 5% non-fat milk diluted in PBS, the membranes were incubated with primary antibody GAPDH, LC3, p62, ZO-1 overnight at 4°C. After being washed, the membranes were incubated with secondary antibody (1: 5000, Proteintech, Wuhan, Hubei, China). Image J software was used for the semi-quantification of protein expression.
Antibodies and reagents
Rabbit anti-ZONAB (bs-12985R, Bioss; 1:100); fluorescent secondary antibody (goat anti-rabbit Alexa 488), Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Life Sciences), rabbit FITC-conjugated antibodies (Proteintech, Wuhan, Hubei, China) the HRP-conjugated secondary antibody (1: 5000, Proteintech, Wuhan, Hubei, China), and Mito-TEMPO (MT; ALX-430-150-M005, Enzo Life Sciences). Rabbit anti-ZO-1 (13663, Cell Signaling; 1:400); rabbit anti-LC3 (Sigma-Aldrich; 1:2000), rabbit anti-p62/SQSTM1 (PM045, MBL; 1:200); rabbit anti-GAPDH (2118, Cell Signaling Technology; 1:10,000); rabbit anti-Occludin (91131, Cell Signaling;1:200); rabbit anti-phospho-Src Family (Tyr416) (6943, Cell Signaling; 1:1000); rabbit anti-Claudin-5 (bsm-52933R, Bioss; 1:500).
RNA isolation and reverse transcription
Total RNA of HBMEC was extracted using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific Life Sciences, Waltham, MA). RNA was further purified from the TRIzol extract with 20% chloroform, centrifuged, and precipitated from the resulting water phase with isopropanol. After RNA extraction, the reverse transcription was synthesized using Revert Aid TM First Strand cDNA Synthesis Kit from Fermentas according to the manufacturer's instructions using random primer.
Real-time polymerase chain reaction
Each reaction contained cDNA. The PCR primers were designed with Primer Premier 5.0 software, and β-Actin was used as a reference gene. qPCR was performed on iQ5 Real-Time PCR Detection System (Bio-Rad, USA) using SYBR Green Real-Time PCR Master Mix (TOYOBO CO., LTD, Japan). P66shc specific primers (Forward: AATTTGGGCCTCTTGTACAGTT, Reverse: TACCTCACAGGCCTAGGCGAGG). ZO-1 specific primers (Forward: CGGTCCTCTGAGCCTGTAAG, Reverse: GGATCTACATGCGACGACAA).
HBMEC was incubated with siRNA targeting p66Shc (sense: 5'-AUGAGUCUCUGUCAUCGCUtt-3'; antisense: 5'-AGCGAUGACAGAGACUCAUtt-3') (GenePharma) for four h in a confocal dish, used Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Life Sciences), followed by growing in standard growth medium in 24h. Scrambled siRNA (sense: 5'-GAUCAUACGUGCGAUCAGA-3'; antisense: 5'-UCUGAUCGCACGUAUGAUC-3' were used as negative controls (NC group).
After being exposed to HG for 24 hours with or without Mito-TEMPO (MT), a mitochondrial ROS inhibitor, cells were grown on L-lysine coated 5% BSA were washed three times with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. After being blocked with 5% BSA for 60 min, cells were incubated with the primary antibodies ZO-1 in the blocking solution at 4℃ overnight. After being washed three times, cells were incubated with a fluorescent secondary antibody (goat anti-rabbit Alexa 488) for ZO-1. After washing, the cell was stained with DAPI. All samples were examined under a fluorescence microscope or confocal microscope.
HBMEC was fixed in 4% PFA/0.1% glutaraldehyde in 100 nm sodium cacodylate, at pH 7.43, dehydrated, and embedded in LR-White resin. We viewed the grids on transmission electron microscope at 80 kV. We identified autophagic vacuoles by a double membrane structure of autophagosomes according to standard criteria.
Cells were treated with DCFH-DA (10μM for 30min at 37°C) in live-cell imaging at 37℃. After washing, the cells were subsequently analyzed by confocal microscopy. The fluorescence intensity was quantified using the open-source cell image analysis software Cell Profiler 63, as described below.
All experiments were performed in triplicate and repeated at least three times. Statistically significant differences between groups were determined using two-tailed one-way ANOVA, followed by a Student–Newman–Keuls test or Student t-test. P < 0.05 was considered statistically significant.