TheHUVECs were purchased from the American Type Culture Collection (ATCC) and incubated with Dulbecco's Modified Eagle Medium (DMEM) (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% Penicillin-Streptomycin Solution (Solarbio, China) at 37 ℃ in 5% CO2 and 95% air.
Cells were cultured to 80~90% confluence and treated for 3, 12, 24 hours with LPS (Sigma. USA) (10 ug/ml) or hydrogen-rich medium (0.6 mmol/L), then the cells were harvested for the following experiments. 25 uM Mdivi-1 (Sigma, USA), a selective inhibitor of Drp1, was joined to the medium 1 h before LPS or hydrogen-rich medium treatment. HUVECs were pretreated for 6 hours with the HO-1 inhibitor zinc protoporphyrin Ⅸ (Znpp) (Sigma, USA) (10 μM).
HUVECs were cultured on 10 mm glass dishes. After stimulation with or without LPS and hydrogen-rich medium. The cell death was tested by Dead Cell Apoptosis Kit with FITC annexin V and PI (Invitrogen, USA) and was performed by following the manufacturer's instruction. The cells were concentrated, and washed twice with cold PBS. Basically, collected individual cells were stained with FITC annexin V and PI, then analyzed by confocal microscope (Nikon). Cytomembrane with bright green fluorescence was apoptotic cell.
HUVECs were cultured on 10 mm glass dishes. After stimulation with or without LPS, hydrogen-rich medium and Znpp, cells were incubated with 500 nM MitoTracker Orange (Molecular Probes-Invitrogen, USA) for 5 min, then washed with PBS, and were examined under confocal microscope (Nikon). HUVECs in 3 independent experiments were used for quantification of mitochondrial aspect ratio (length/width) using ImageJ-3D Object counter plug-in. Each experiment was done at least four times and each time 16–25 cells per condition were quantified. Aspect ratio was defined as length/width.
The rates of oxygen consumption in cybrid cell lines were measured with a Seahorse Bioscience XF-24 extracellular flux analyzer (Seahorse Bioscience) [26, 27]. XF24 creates a transient, 7 ul-chamber in specialized microplates that allows for the determination of oxygen and proton concentrations in real time. To allow comparison between different experiments, data are expressed as the rate of oxygen consumption in pmol/min. Normalized to cell protein in individual wells determined by the Bradford protein assay (Bio-Rad). A density of 60 000 cells per well in 24-well plate was coated with Cell-TakTM adhesive. The rates of O2 were determined under basal condition and the addition of oligomycin (1.0 uM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.3 uM), rotenone (1.0 uM) and antimycin A (1uM) [26, 27].
Mitochondrial membrane potential
HUVECs were cultured on 10 mm glass dishes. After stimulation with LPS, cells were incubated with 500 nM JC-1 for 20 min (Biyuntian, Nanjng, China), washed with PBS, and were examined under confocal microscope (Nikon). HUVECs in 3 independent experiments were used for quantification. Each experiment was done at least four timess.
Western blot analysis
Equal amounts of protein from cells were separated by SDS–PAGE (10% polyacrylamide gels) and electrotransferred to nitrocellulose. Membranes were blocked with 5% defatted milk in Tris-buffered saline, pH 7.6, containing 0.1% (v/v) Tween 20 (TBST). Membranes were incubated with primary antibodies at 4 ℃, total Drp1 1:1000 (Cell Signalling, USA), HO-1 1:1000 (Abcam, USA), β-actin 1:1000 (Sigma, USA) and re-blotted with horseradish peroxidase-linked secondary antibody 1:5000 (Proteintech Group, USA). The bands were detected using ECL (Perkin Elmer, USA) with exposure to Kodak film and quantified by scanning densitometry. Protein content was normalized by β-actin.
Drp1 and mitochondria colocalization
HUVEC cells cultured on 10 mm glass dishes were stimulated with LPS for 12h. During the last 5 min of treatment 500 nM MTO was added. Cells were washed with PBS and fixed with PBS containing 4% paraformaldehyde and incubated for 10 min in ice-cold. 0.3% Triton X-100 (Sigma–Aldrich, USA) were used for 10 min for permeabilization. Nonspecific sites were blocked with 5% Goat Serum in PBS for 1 h and then the cells were incubated with anti-Drp1 (1:50) antibody (Cell Signalling, USA). Secondary antibody was anti-rabbit (Molecular Probes-Invitrogen, USA). For the colocalization analysis only one focal plane was analyzed with a Zeiss LSM-5 Pascal 5 Axiovert 200 microscope. Images obtained were deconvolved and background was subtracted using the ImageJ software. Colocalization between the Drp1 and mitochondria was quantified using the Manders’ algorithm, as detailed elsewhere [28, 29].
Data are shown as mean ± SD (standard deviation). Data were analyzed by one-way ANOVA and differences among groups was detected using a Dunnett’s test. Statistical significance was defined as P < 0.05.