CM-H₂-DCFDA, JC-1, rotenone, antimycin-A and both reduced and non-reduced Mito Tracker were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The antibodies against PHB1 and cyclooxygenase (COX) IV were purchased from Abcam, while the Annexin V-FITC apoptosis kit was obtained from Becton, Dickinson and Company. The antibodies against β-actin, cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase-3 were obtained from Cell Signaling Technology, Inc. and DAPI from MilliporeSigma.

HRCECs, obtained from the BeNa Culture Collection (Shanghai, China), were cultured in Endothelial Cell Medium (ECM; ScienCell Research Laboratories) supplemented with 5% FBS (Gibco; Thermo Fisher Scientific, Inc.) in 5% CO₂. The cells were used for subsequent experiments after 4-5 passages. When cells reached 50-60% confluence, they were treated with 5.5 mmol/l [normal glucose (NG)] or 30 mmol/l glucose (HG) for 4-6 days. Subsequently, the cells were divided into the following four groups: The NG group; the HG group; the HG + negative control (NC) group; and the HG + PHB-transfected group.

HRCECs were first cultured for 24 h and were then transfected with the pCMV6-XL5 plasmid (Clontech) encompassing the PHB sequence. When cells reached 50-60% confluency, they were transfected with the PHB overexpression plasmid (0.2 µg) using a X-tremeGENE HP DNA transfection reagent (Roche Applied Science) at 37˚C for 24 h according to the manufacturer's instructions. Following transfection for 24 h the medium was replaced with normal medium supplemented or not with HG (30 mmol/l). The transfection efficiency was ~70-80%.

HRCECs were cultured in ECM supplemented with 5% FBS at 37˚C and 5% CO₂ for 24 h. When cells reached 50-60% confluency, they were transfected with small interfering RNA (siRNA, 50 nM) clones against PHB (Qiagen) using HiPerFect transfection reagent (Qiagen) for 10 h at 37˚C according to the manufacturer's instructions. Cells were collected at 48 h after transfection for various assays. The sequence of PHB-siRNA: 5'-AATGTGGATGCTGGGCACAGA-3'; the sequence of negative control: 5'-AAGAGTGTCGGATGCAGGATC-3' (Qiagen).

The subcellular fractions of HRCECs were separated as previously described (9). Briefly, HRCECs were rinsed in PBS and were then resuspended into Buffer A (20 mmol/l HEPES pH 7.5, 10 mmol/l KCl, 1.5 mmol/l MgCl2, 1 mmol/l EGTA, 1 mmol/l EDTA, 1 mmol/l DTT, 0.1 mmol/l PMSF, 250 mmol/l sucrose) supplemented with protease inhibitors. The cells were homogenized by 10 strokes in a Dounce homogenizer. Subsequently, cells were centrifuged at 1,000 x g for 4 min at 4˚C to isolate cell nuclei and DNA fragments. The supernatant was then collected and centrifuged at 8,000 x g for 20 min at 4˚C to isolate heavy cellular fragments rich in mitochondria. The resultant supernatant was centrifuged at 10,000 x g for 15 min at 4˚C to obtain the cytosolic fractions.

Western blot analysis was performed as previously described (10). Briefly, equal amounts of protein (20 µg) were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Sigma). The membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in Tris-buffered saline Tween-20 (0.1%) for 2 h at room temperature and then incubated with primary antibodies at 4˚C overnight. The following primary antibodies were used: Anti-PHB (dilution, 1:500; product code ab75766; Abcam), anti-COX IV (dilution, 1:700; product code ab202554; Abcam), anti-cleaved-caspase 3 (dilution, 1:400; product no. 9661; Cell Signaling Technology, Inc.), anti-cleaved PARP (dilution, 1:500; product no. 5625; Cell Signaling Technology, Inc.) and anti-β-actin (dilution, 1:1,000; cat. no. sc-8432; Santa Cruz Biotechnology, Inc.). Immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.). Quantification was performed according to the NIH Image version-1.61 software (National Institutes of Health).

HRCECs were cultured on coverslips and were then fixed with 4% paraformaldehyde for 10 min at room temperature and 0.2% Triton X-100. Subsequently, cells were blocked with PBS for 1 h at room temperature supplemented with 0.2% Tween-20, 10% BSA and 10% serum (Gibco; Thermo Fisher Scientific, Inc.). Following blocking, the cells were first incubated with the indicated primary antibody (PHB; dilution, 1:100; product code ab75766; Abcam) and then with Alexa Fluor 488-conjugated secondary antibody (dilution, 1:1,000; product code ab150077; Abcam). The cell nuclei were stained for 5 min at room temperature with 2 µg/ml DAPI. For Mito Tracker staining, HRCECs were stained for 20 min at room temperature with 0.02 µM Mito Tracker (Invitrogen; Thermo Fisher Scientific, Inc.). For JC-1 staining, HRCECs were rinsed with PBS and incubated with JC-1 (Invitrogen) staining solution at 37˚C for 20 min. Pictures were obtained with an FV-1000 confocal laser scanning microscope (Olympus). For live cell imaging, images were captured under a phase contrast microscope (Carl Zeiss GmbH).

Following HRCEC trypsinization, cells (1x10⁶/ml) were resuspended and oxygen consumption was measured using an oxygen probe in the presence or absence of 10 µM rotenone (an inhibitor of complex I) 10 µM antimycin-A (an inhibitor of complex III) and 0.05% azide, an inhibitor of mitochondrial respiration. Finally, the activity of complex I/III was compared between PHB-knocked down and control cells.

Firstly, HRCECs (1x10⁶/ml) cultured in DMEM supplemented with 0.2% Fetal Calf Serum (GIBCO) were incubated with 5 µM CM-H₂-DCDA and 5 µg/ml propidium iodide (PI) at room temperature for 20 min. HRCECs treated with 100 µM H₂O₂ for 20 min served as a positive control group. H₂O₂ levels were detected using CM-H₂-DCDA. Subsequently, PHB-depleted cells were treated with 10 µg/ml PEG-catalase for 2 h at room temperature. Living cells were analyzed using PI staining for 10 min at room temperature and the fluorescence intensity in the FL-2 and FL-1 channels was measured using a Becton-Dickinson FACSCalibur system (BD Biosciences) and FlowJo 7.6 software (Tree Star, Inc.).

Flow cytometric analysis was performed as previously described (10). In brief, cells (1x10⁶/ml) were used to assess cell apoptosis with an Annexin V/propidium iodide (PI) kit (Invitrogen, Thermo Fisher Scientific, Inc.) according the manufacturer's protocol. Flow cytometry was used determine the proportion of early apoptotic cells. Flow cytometry was performed using a FACSCalibur system (BD Biosciences).

Experiments were performed at least three times. All data are presented as the mean ± SD. Differences between multiple groups were assessed using a post hoc (Tukey's HSD) test used for one-way ANOVA in GraphPad Prism 6.0 (GraphPad Software, Inc.) and SPSS 17.0 (SPSS, Inc.). Comparison between non-parametric variables was performed using a χ² test. P<0.05 was considered to indicate a statistically significant difference.

To detect the localization of PHB, immunofluorescence analysis was carried out in HRCECs. The analysis revealed that a small proportion of PHB was distributed in the nuclear compartment, while the majority of PHB was detected in the mitochondria, as shown using Mito Tracker (Fig. 1A). Furthermore, to determine the expression levels of PHB in the mitochondria under HG conditions, HRCECs were exposed to 30 mmol/l glucose (HG). The results revealed that PHB was downregulated in the mitochondria of HRCECs in the HG group (Fig. 1B and C). In addition, the expression levels of PHB were measured in all the different groups and the results demonstrated that PHB was successfully overexpressed in cells transfected with PHB overexpression plasmid (Fig. 1D and E). Collectively, these findings indicated that PHB could be involved in the regulation of mitochondrial function in HRCECs.

To investigate the role of PHB in mitochondria, PHB-depleted mitochondria were established in HRCECs and the levels of ROS were then measured. Immunofluorescence staining in HRCECs revealed reduced Mito Tracker positive staining, indicating that mitochondria were inactive with regard to respiration (Fig. 2A). This finding clearly suggested that the increased ROS production in PHB-depleted HRCECs was due to mitochondrial respiration. Furthermore, both immunofluorescence (Fig. 2B) and ROS production (Fig. 2C and D) analyses revealed reduced red and enhanced green fluorescence signals in PHB-depleted HRCECs, thus suggesting that mitochondrial membrane depolarization was promoted and ROS production was increased, respectively. In addition, the activity of complex I/III was assessed via measuring oxygen consumption. The results demonstrated that the activity of complex I was reduced in PHB-depleted HRCECs compared with the control group (Fig. 2E). However, the activity of complex III remained unchanged between both groups (Fig. 2F). Overall, the aforementioned results verified that mitochondria mediated ROS generation in PHB-depleted HRCECs.

To explore whether PHB could protect HRCECs, the effect of PHB on HG-induced cell apoptosis was assessed by evaluating the apoptosis-specific morphological changes in HRCECs. Therefore, HG markedly enhanced HRCEC apoptosis, which was restored by PHB (Fig. 3A). Additionally, HRCEC apoptosis was determined by Annexin V/PI staining. The apoptosis rate in the NG group was 9.96%, which was significantly different compared with that in the HG, NC or PHB groups, with apoptosis rates of 29.40, 23.64 and 12.06%, respectively (Fig. 3B and C). Collectively, the aforementioned findings suggested that PHB could attenuate HG-induced HRCEC apoptosis.

Subsequently, the effect of PHB on the expression levels of the apoptosis-related proteins, PARP and caspase-3, in NG-, HG-, NC- and PHB-treated cells was evaluated by western blot analysis. Consistent with the Annexin V/PI staining results, western blot analysis revealed that the expression levels of both caspase-3 and PARP were significantly increased in the HG and NC groups compared with NG group. However, the expression levels of both proteins were not notably different between the HG and NC groups. Notably, caspase-3 and PARP were markedly downregulated in the PHB group (Fig. 4A-C). Collectively, the aforementioned findings suggested that PHB could protect HRCECs against HG-induced apoptosis via downregulating caspase-3 and PARP.