Cell culture and survival.
Human umbilical vein endothelial cells purchased from (ATCC), and cultured in EGM-2 bullet kit system (#CC-4147, Lonza), containing endothelial basal medium 2 (#CC-3156, Lonza), 5% fetal bovine serum (FBS), human vascular endothelial growth factor (hVEGF), human basic fibroblast growth factor (b-FGF), human epidermal growth factor (EGF), human insulin-like growth factor-1 (IGF-1), and ascorbic acid. FUNDC1 silence or overexpressed cells were treated with and without high glucose and cell survival was detected using D-Plus cell counting kit (CCK-8) according to the manufacturer’s instructions (#CCK-3000, Dongin Biotech). Followed by transfection or treatment cells were seeded into 96 well plates and incubated overnight at 37oC in a 5% CO2 incubator. Next, cells were treated with (10 µL/well) WST-1 for 1 h in the dark at 37oC in a 5% CO2 incubator. The absorbance of each well was measured at 450 nm using a Tecan (XFluor, Switzerland) micro plate reader. Each experiment were repeated 3–6 times.
Myc-FUNDC1 transfection.
Endothelial cells were transfected with 1.5 µg/ml of Myc-FUNDC1 using Megatrans 2.0 transfection reagent according to the manufacture’s protocol. Post three hours of transfection the media containing the transfection reagent was replaced by fresh media and further experiments were performed.
siRNA transfection.
Endothelial cells were transfected with FUNDC1 SiRNA, targeting humans in serum reduced OPTI-MEM media. The cells were transfected with Lipofectamine RNAiMax transfection reagent based on the manufacturer’s instructions. Following, four hours of incubation with transfection reagent, media containing transfection reagents were removed and added fresh media with and without high glucose and the further experiment was performed.
Tube formation assay.
Growth factor reduced matrigel (#354230, CORNING) was used for this experiment. GFR-Matrigel was thawed at 4°C in a day before the experiment and then 96 well plates were coated with matrigel (60 µL/well) and polymerized for 30–45 min at 37°C in a CO2 incubator. FUNDC1 overexpressed cells were maintained with and without high glucose for 48 hours. Then, cells were labeled with MitoTracker® Green FM (#M7514, Life Technologies) 200 nM, and LysoTracker deep red (#12492, Invitrogen) 200nM for 30 minutes at room temperature in the dark. MitoTracker and LysoTracker labelled cells were collected and seeded on matrigel-coated wells at a density of 6×103 cells/well in the full medium. After 6 h incubation at 37°C in a CO2 incubator, the branch formation was captured in Lion Heart FX automated microscope (Biotek, USA) at 4x magnification with different microscopic channels. The branch formation was quantified using ImageJ software.
Annexin V FITC/PI staining.
Flow cytometer analysis was performed to quantify the mitochondrial apoptosis by FITC Annexin V apoptosis detection kit (#556547, BD pharmingen). FUNDC1 overexpressed or silenced cells were collected and seeded on six-well plates and incubated with and without high glucose for 48 hours at 37oC in a CO2 incubator. Post treatment the cells were stained with Annexin V FITC and PI for 15 minutes at room temperature in the dark and analyzed by FACS (BD Facs canto II, San Jose, CA, USA). Annexin V FITC negative and PI negative cells are viable cells whereas the duplet positive cells were apoptotic cells.
Immunoblots.
Whole-cell lysates were extracted using Pierce RIPA buffer (#89901, Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktail, and protein concentration was determined by BCA assay kit. Protein samples were separated with SDS-PAGE (6–15% gel) and transferred to PVDF membranes (#IPVH00010, Immobilon-P-Millipore). The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies, FUNDC1 (#49240), Bax (#2772S), Cleaved caspase 3 (9661S), LC-3 A/B (#12741S), phosphor form of AKT, P70S6K, 4E-BP1 and total form of AKT, P70S6K, and 4E-BP1 from Cell Signaling technology. FUNDC1 (#bs-3227R), from Bioss. TOMM20 (#sc-17764), Beta-Actin (#sc-47778) from Santa Cruz Biotechnology. p62 (#NBP1-48320), LA-3B (#NB100-2220) from Novusbio, and DDK (#TA50011) from ORIGENE for overnight at 4°C. Followed by incubation with HRP conjugated Goat anti-rabbit IgG (#ADI-SAB-300-J) and Goat anti-mouse IgG (#ADI-SAB-100-J) from Enzo Life Sciences secondary antibodies at 25°C for 1 h and western blots were perceived using chemiluminescent detection reagent (#WBLUR0500, Immobilon, Millipore).
In vitro immunofluorescence staining.
For, immunofluorescence staining endothelial cells were allowed to grow on Poly-L-lysine (#P4707, Sigma) coated coverslip. Once the cells reached 70–80% level of confluence transfected with FUNDC1. Transfected cells were exposed with and without high glucose for 48 hours and labeled with MitoTracker® deep red (#M22426, Invitrogen) 200 nM for 30–60 minutes. The cells were then fixed with 4% PFA or 100% cold methanol for 5–10 min at 25°C. Cells permeabilized with 0.1% Triton x-100 in 1x PBS for 10 min and blocked with 1% BSA in 0.3 M glycine for 1 h. Further, cells stained with primary antibodies against LAMP2 (#sc-18822), Tom20 (#sc-17764) from Santa Cruz Biotechnology, and LC-3B (#NB100-2220) from Novusbio for overnight at 4°C in the dark. After washing thrice with PBS (each wash 5 minutes), and incubated with Alexa flour conjugated secondary antibodies (#A11001) from Invitrogen for 1 hour at room temperature in the dark. Then cells were covered with a coverslip using vectashield vibrance mounting medium (#H-1700, VECTOR laboratories) and images were captured using Lion Heart FX automated microscope (Biotek, USA) at 40x magnification. Scale bar (100 µm).
Reverse Transcription-PCR.
RT-PCR was conducted to examine the effects of BAM15 on HG, and NG stimulated cells. Total RNA was purified by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Then RNA (1.0 lg) was reverse transcribed using the M-MLV reverse transcriptase to synthesize complementary DNA. The reverse transcription was performed according to the manufacturer’s instructions from PrimeScript 1st strand cDNA synthesis kit from TAKARA. Polymerase chain reaction was performed for amplification of complementary DNA using specific primers. Then PCR was performed using the following cycle parameter, the denaturation at 94 C for 30 s, annealing at 59 C for 30 s, and extension at 72 C for 30 s. After 40 cycles, amplification products were electrophoresed on 1% agarose gel visualized by EtBr staining. Then, UV trans illuminator was used to photograph and analyze the gray value of the mRNA expression in each group.
Mitochondrial membrane potential (TMRE) assay.
Mitochondrial membrane polarization was detected using TMRE probe dye. FUNDC1 silenced or overexpressed cells were exposed to high glucose and loaded with TMRE probe for 30 minutes at 37oC in a culture medium. Following incubation with TMRE probe, cells were washed with PBS, and images were captured with RFP channels in Lion Heart FX automated microscope (Biotek, USA) with 10x magnification, and mitochondrial depolarization was determined by a decrease in the RFP/GFP fluorescence intensity ratio. Mitochondrial polarization quantified by Gen5 software in Lion Heart FX automated microscope (Biotek, USA).
Detection of cellular and mitochondrial ROS.
Cellular and mitochondrial ROS level measured using cellROX green (#C10444, Invitrogen), mitoSOX red reagent respectively. Briefly, endothelial cells followed by HG or BAM15 treatment, media were replaced with fresh media containing 5 µM of cellROX green reagent or mitoSOX red reagent and incubated for 15 minutes at 37oC. After that, flow cytometer analysis was carried out by fluorescence-activated cell sorting (FACS; BD FACS canto 2, San Joes, CA, USA). Fluorescence-activated cell sorting was performed by using non-stained cells as a negative control. The fraction of positively stained cells was determined by comparison with non-stained cells.
Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
Mitochondrial respiration was evaluated by quantifying the OCR or ECAR using a Seahorse XF extracellular flux analyzer. Briefly, endothelial cells treated by HG or BAM15 have used this experiment. The 8000 cells/well in cell culture mini plates were incubated at 37oC in a CO2 incubator for the day before assay. On the day of the experiment, assay medium was prepared and supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. OCR and ECAR rate analyzed using 2 µM oligomycin, 4 µM FCCP, and 1 µM rotenone, and 1 µM antimycin A as indicated. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a seahorse XFp analyzer.
Statistical analysis.
Statistical analyses were performed using GraphPad Prism software (Version 5) and one-way analysis of variance (ANOVA) and Student T-test (Paired) was used to assess the differences between experimental and control groups. Data are presented as mean ± standard error of the mean (SEM). The results were considered statistically significant at P < 0.05 (*). P values less than 0.01 or 0.001 were indicated with ** or ***, respectively. The experimental data presented was an average of three independent experiments.