The mouse eNOS cDNA (BC052636.1) was cloned into the eukaryotic expression vector pcDNA3.0 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using NotI and HindIII restriction sites. The forward primer was 5′-ATAAGAATGCGGCCGCATGGGCAACTTGAAGAGTGTGG-3′ (NotI site underlined) and the reverse primer was 5′-CCCAAGCTTTCAGGAACCAGGTGTTTCTTGGG-3′ (HindIII site underlined). Subsequently, the recombinant vector was amplified in DH5α Escherichia coli (Sangon Biotech Co., Ltd., Shanghai, China) and plasmid DNA was purified with an endotoxin-free plasmid purification kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. Each segment was amplified by polymerase chain reaction (PCR) with Takara LA Taq or Primestar (Takara Bio Inc., Otsu, Japan) and cloned into the pcDNA3.0 vector. Furthermore, all joints in the constructs were confirmed by sequencing (Sangon Biotech Co., Ltd.). The present study was approved by the Ethics Committee of Guilin Medical University (Guilin, China).

The mouse pancreatic β cell line Min6 (American Type Culture Collection, Manassas, VA, USA) was maintained in Dulbecco's modified Eagle's medium with high glucose, 10% heat inactivated newborn calf serum, and 1% antibiotic-antimycotic solution (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Min6 cells were treated with pcDNA3.0-eNOS plasmid (1 µg) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 24, 48, 72 and 96 h. Empty pcDNA3.0 vector (1 µg) was used as a negative control.

Min6 cells were seeded at a density of 1.0×10⁵ cells/ml in 6-well plates in order to achieve ~50% confluence the next day, and were then transfected with pcDNA3.0-eNOS (50 µM). Thereafter, 100 µl cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) solution was added to each well and were incubated with the cells for 1 h. The absorbance was then measured at 450 nm using a microplate reader.

Following plasmid transfection for 24 h, the apoptotic cells were quantified using an Annexin V/propidium iodide (PI) apoptosis kit (Multi Sciences Biotech Co., Ltd., Hangzhou, China). Min6 cells were collected, washed with PBS and resuspended in 200 µl binding buffer containing 5 µl Annexin V (10 µg/ml) for 10 min in the dark. The cells were then incubated with 10 µl PI (20 µg/ml), and the samples were immediately analyzed using flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). Data acquisition and analysis was performed using CellQuest software (CellQuest Pro, version 5.1; BD Biosciences, Franklin Lakes, NJ, USA).

Total RNA was isolated using a UNIQ-10 column and TRIzol total RNA isolation kit (Sangon Biotech Co., Ltd.). In total, 3 µg total RNA was used for reverse transcription in a reaction volume of 20 µl using Cloned AMV Reverse Transcriptase (Invitrogen; Invitrogen; Thermo Fisher Scientific, Inc.). In addition, 3 µl cDNA was used for qPCR using a Takara Ex Taq RT-PCR version 2.1 kit (Takara Bio, Inc.). Gene-specific PCR primers for eNOS, SIRT1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are listed in Table I, and PCR signals were detected with a DNA Engine Opticon 2 Continuous Fluorescence Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). PCR was monitored for 45 cycles using an annealing temperature of 60°C. At the end of the PCR cycles, melt curve analysis and 2% agar electrophoresis was performed in order to assess the purity of the PCR products. Negative control reactions (no template) were routinely included to monitor potential contamination of reagents. Relative quantities of eNOS mRNA and SIRT1 mRNA were normalized to the quantity of GAPDH mRNA using the 2^−∆∆Cq method (13).

The concentration of protein in extracts from mice pancreatic β cells was determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein lysates were prepared using NP-40 buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) on ice for 20 min before centrifuging at 30, 000 × g for 30 min at 4°C. The supernatant was subsequently separated by 10% SDS-PAGE (20 µg protein/lane) followed by transfer onto nitrocellulose membranes. Western blot analysis was performed as previously described (14), and the signals were detected using an enhanced chemiluminescence (ECL) system (Millipore, Billerica, MA, USA). Antibodies used in the present study included anti-mouse eNOS (cat. no. SA-201-0100; 1:4,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-mouse SIRT1 (cat. no. sc-74465; 1:4,000; Santa Cruz Biotechnology, Inc.) and anti-mouse GAPDH (cat. no. sc-365062; 1:20,000; Santa Cruz Biotechnology, Inc.). The blots were subsequently incubated with a secondary antibody (horseradish peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG; cat. no. 111-035-003; 1:10,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at room temperature for 2 h. and exposed to ECL reagent according to the manufacturer's protocol for the detection of protein expression.

Protein-protein interactions were analyzed by co-IP experiments. The cells were collected, and the proteins were solubilized in IP buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% protease inhibitor mixture]. Co-IP was performed according to the standard protocols previously described (15,16). Briefly, the Min6 cells were washed with ice-cold PBS twice and lysed in ice-cold radioimmunoprecipitation assay buffer (cat. no. P0013B; Beyotime Institute of Biotechnology, Haimen, China). The supernatant was incubated with 10 µl mouse anti-eNOS (cat. no. SA-201-0100; 1:100; Santa Cruz Biotechnology, Inc.) or mouse anti-SIRT1 (cat. no. sc-74465; 1:100; Santa Cruz Biotechnology, Inc.) monoclonal antibody and agarose ligand (Catch and Release v2.0; EMD Millipore, Billerica, MA, USA), followed by incubation at 4°C for 1 h. The immune complexes were washed, eluted by boiling in 2X SDS sample buffer, separated by 10% SDS-PAGE and transferred onto membranes. The blots were then incubated with a horseradish-peroxidase-conjugated secondary antibody (cat. no. BHR101-1; 1:10,000; Bersee, Biomart company, Beijing, China) at room temperature for 1 h and exposed to ECL reagent for the detection of protein expression according to the manufacturer's protocol.

Differences between each group were expressed as the mean ± standard deviation. Statistical significance was assessed by Student's t-test and one-way analysis of variance followed by a Tukey post hoc test. P<0.05 was considered to indicate a statistically significant difference.

eNOS expression was upregulated following pcDNA3.0-eNOS transfection (Fig. 1). The mRNA (P<0.001; Fig. 1A) and protein (Fig. 1B) levels of eNOS in Min6 cells were clearly increased following pcDNA3.0-eNOS transfection for 24 h.

In order to determine the effect of eNOS on SIRT1, SIRT1 expression at the mRNA and protein levels was detected, as shown in Fig. 2. The mRNA (P<0.001; Fig. 2A) and protein (Fig. 2B) levels in Min6 cells were clearly upregulated following transfection with pcDNA3.0-eNOS for 24 h.

The effects of eNOS overexpression on Min6 cell proliferation were examined. As shown in the CCK-8 assay results in Fig. 3, the cellular population was increased time-dependently in the pcDNA3.0-eNOS transfection group compared with the negative control (pcDNA3.0) group, particularly at 48 h (P<0.01) and 72 h (P<0.001).

Cell apoptosis was analyzed using flow cytometry following pcDNA3.0-eNOS transfection for 24 h based on the CCK-8 results. Exposure of Min6 cells to the recombinant eNOS plasmid inhibited the apoptosis of the cells compared with that in the negative control group. Furthermore, the occurrence of apoptosis was significantly lower (P<0.001) in the pcDNA3.0-eNOS group compared with the pcDNA3.0 group (negative control), as shown in Fig. 4.

Finally, the possibility that eNOS interacts with SIRT1 was investigated. To this end, Min6 cell lysates were harvested, and then were subjected to Co-IP and the results in Fig. 5 clearly indicated that there is an interaction between exogenous SIRT1 and eNOS proteins.