MC3T3-E1 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and were plated in 6-well plates at a density of 1.5×10⁶ cells/well in Minimum Essential α-Medium (αMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 10 units/ml penicillin and 10 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). For the SIRT1 silencing experiment, transfection of MC3T3-E1 cells was performed by using small interference (si)RNA. The siRNA sequences that were used were as follows: si-SIRT1 sense, 5′-GAGACUGCGAUGUCAUAAUTT-3′ and antisense, 5′-AUUAUGACAUCGCAGUCUCTT-3′; and scrambled siRNA sense, 5′-CGCUCCGAACGUGCUACGUTT-3′ and antisense, 5′-ACGGUACACGUUCAAAGAATT-3′. siRNAs were purchased from GE Dharmacon (Lafayette, CO, USA). Transient transfection was performed using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h using a final concentration of siRNAs of 5 nM, according to the manufacturer's protocol). For the SIRT1 overexpression experiment, MC3T3-E1 cells were transfected for 8 h at 37°C with 1×10⁹ particle forming units of SIRT1 adenovirus, which was obtained from Professor B.H. Park (Department of Biochemistry, Chonbuk National University, Jeonju, Republic of Korea). Viruses were purified from the supernatants of 293 cell cultures by cesium chloride density gradient centrifugation at 70,000 × g at 4°C for 3 h. As a control, the pAd/CMV/V5-GW/lacZ vector (Invitrogen; Thermo Fisher Scientific, Inc.) was used to produce LacZ-bearing adenovirus. MC3T3-E1 cells were incubated in α-MEM medium without antibiotics for 24 h prior to transfection to enhance the transfection efficiency.

MC3T3-E1 cells were incubated at 37°C in anaerobic jars (Oxoid; Thermo Fisher Scientific, Inc.) with oxygen-absorbing packs (AnaeroGen; Oxoid; Thermo Fisher Scientific, Inc.). Prior to transferring to the hypoxic chamber, the cells were moved to a glucose-free medium. Within 0.5 h, O2 had decreased to less than 0.1%.

An MTT assay was used to estimate cell viability (12,13). Briefly, cells were plated at a density of 1×10⁴ cells/well in α-MEM medium supplemented with 10% FBS and antibiotics in 96-well plates. Following exposure to hypoxia for 24 h, MTT solution (Sigma-Aldrich; Merck KGaA) was added in plates at a final concentration of 0.5 mg/ml for 3 h at 37°C. Following removal of the medium, 100 µl dimethyl sulfoxide solution was added to dissolve the formazan crystals. The absorbance at 570 nm was detected with a microplate reader (Synergy 4 Hybrid Multi-Mode; BioTek Instruments, Inc., Winooski, VT, USA).

Caspase 3 and 7 activity was detected using a luminescence-based Caspase-Glo^® 3/7 Assay (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Cells were cultured in hypoxia for 24 h and Caspase-Glo 3/7 reagent was added to each well in a 1:1 ratio according to the manufacturer's protocol. Following agitation for 10 min on a plate shaker at room temperature, 90% of the lysate volume was transferred to a 96-well solid-white plate. Cell lysates were analyzed with a microplate reader (Synergy 4 Hybrid Multi-Mode; BioTek Instruments, Inc.).

Hypoxia-induced cell apoptosis was determined using the Cell-APOPercentage™ Apoptosis Assay (cat. no. GB2356929; Biocolor Ltd., Carrickfergus, Northern Ireland). Digital images of APOPercentage dye-labeled cells, appearing bright pink against a white background under a light microscope, were used to detect apoptotic cells. To quantify the level of apoptosis, cells were incubated with a 1:100 dilution of the dye for 5 min at 37°C following culture under each experimental condition. Cells were then washed with PBS and the dye within the labeled cells was released into the supplied dye-release reagent. The contents of each well (250 µl) were transferred to a 96-well flat-bottomed plate, and absorbance was read at 550 nm with a microplate reader.

The relative levels of SIRT1, tumor necrosis factor receptor-associated factor 2 (TRAF2) and cellular inhibitor of apoptosis 2 (cIAP2) mRNA were measured using RT-qPCR, as previously described (13). Total RNA was extracted from cells at a density of 1.5×10⁶ cells/well, using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was precipitated with RNase-free chloroform and isopropanol (Sigma-Aldrich; Merck KGaA) and dissolved in diethyl pyrocarbonate-treated distilled water (Promega Corporation). Total RNA (1 µg) free of genomic DNA contamination was reverse-transcribed into cDNA using the random hexamer primer provided in the RevertAid First Strand cDNA Synthesis kit (cat. no. K1612; Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol in a 20 µl reaction volume. The temperature protocol was as follows: At 25°C for 5 min, at 37°C for 60 min and at 70°C for 5 min. Specific primers for each gene were designed using the Primer Express software version 3.0 (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primer sequences used in the present study were as follows: SIRT1 forward, 5′-AAGTTGACTGTGAAGCTGTACG-3′ and reverse, 5′-TGCTACTGGTCTTACTTTGAGGG-3′; TRAF2 forward, 5′-TCCCTGGAGTTGCTACAGC-3′ and reverse, 5′-AGGCGGAGCACAGGTACTT-3′; cIAP2 forward, 5′-TTTCCGTGGCTCTTATTCAAACT-3′ and reverse, 5′-GCACAGTGGTAGGAACTTCTCAT-3′; and GAPDH forward, 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′. qPCR was performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR reactions were carried out in a final volume of 25 µl, containing 1 µl forward and 1 µl reverse primers (10 µM), 0.5 µl cDNA (500 nM), 0.5 µl dNTP Mix (200 µM), and 1 U (0.2 µl) GoTaq^® DNA polymerase in 1X Green GoTaq^® Reaction Buffer (Promega Corporation). Thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing for 45 sec at an optimised temperature for each primer pair, and extension at 72°C for 90 sec, according to the manufacturer's protocol. Relative gene expression was quantified according to the comparative Cq method (14) and normalized to GAPDH expression. All reactions were performed in triplicate.

Cells (1.5×10⁶) were homogenized and lysed in lysis buffer (20 mmol/l HEPES, pH 7.2, 1% Triton X-100, 10% glycerol, 1 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin; Sigma-Aldrich; Merck KGaA) on ice for 30 min. Protein concentrations were quantified using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts (20 µg) of extracted protein samples were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Following blocking in 5% skimmed milk at 4°C for 1 h, the membranes were probed with the following primary antibodies at 4°C for 12 h: Anti-SIRT1 (1:1,000; cat. no. 8469; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phosphorylated (p)-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), anti-acetylated-lysine (1:1,000; cat. no. 9441; Cell Signaling Technology, Inc.), anti-β-actin (1:2,000; cat. no. sc-81178; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-B-cell lymphoma 2 (Bcl-2; 1:1,000; cat. no. sc-492; Santa Cruz Biotechnology, Inc.), anti-Bcl-2 associated X protein (Bax; 1:1,000; cat. no. sc-20067; Santa Cruz Biotechnology, Inc.), anti-caspase 3 (1:1,000; cat. no. sc-1225; Santa Cruz Biotechnology, Inc.), anti-caspase 9 (1:1,000; cat. no. sc-133109; Santa Cruz Biotechnology, Inc.), anti-cleaved-caspase 3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.), anti-cleaved caspase 9 (1:1,000; cat. no. 9509; Cell Signaling Technology, Inc.), anti-p-Akt (1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), and anti-hypoxia inducible factor-1α (HIF-1α; 1:2,000; cat. no. ab113642; Abcam, Cambridge, MA, USA). Membranes were then incubated with the following horseradish peroxidase-conjugated secondary antibodies; goat anti-mouse immunoglobulin (Ig) G (1:5,000; cat. no. 31430; Thermo Fisher Scientific, Inc.) and goat anti-rabbit IgG (1:5,000; cat. no. 31460; Thermo Fisher Scientific, Inc.) at 4°C for 1 h. Protein bands were visualized using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) on an Amersham Imager 600UV system (GE Healthcare Life Sciences, Little Chalfont, UK).

Whole cell lysates were pre-cleared with Protein A/G plus (Santa Cruz Biotechnology, Inc.) for 30 min at 4°C. Beads were pelleted at 1,000 × g for 30 sec at room temperature, and pre-cleared supernatants were incubated with 10–20 µg primary antibodies against SIRT1 (1:50; cat. no. 8469; Cell Signaling Technology, Inc.) and Akt (1:50; cat. no. sc-5298; Santa Cruz Biotechnology, Inc.), using normal magnetic bead-conjugated IgG (1:50; cat. no. 5873; Cell Signaling Technology, Inc.) as a negative control. The reactions were kept on a rotating wheel at 4°C overnight. When agarose or a gel conjugate was unavailable, lysates were incubated with primary antibody or an equivalent amount of control IgG for 2 h at 4°C and then overnight along with Protein A/G plus beads to collect the immune complexes. Beads were collected by centrifugation at 16,000 × g at 4°C for 10 min, washed several times with radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc.), washed once with PBS, and re-suspended in SDS-PAGE sample loading buffer (Geno Technology, Inc., St. Louis, MO, USA). Immune complexes and 75–100 µg input proteins were resolved by 12% SDS-PAGE.

Data are expressed as the mean ± standard error of the mean of at least 3 independent experiments. The statistical significance of the differences between groups was assessed using one-way analysis of variance followed by a post hoc Fisher's protected least significant difference test for multiple comparisons. Statistical analysis was performed using GraphPad Prism software version 5.02 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR and western blot analysis were used to detect the expression level of SIRT1 in hypoxia-induced apoptosis in MC3T3-E1 cells. The differences in mRNA expression were compared with RT-qPCR at different times under hypoxia. As demonstrated in Fig. 1A, SIRT1 mRNA appeared to be elevated in the first 12 h of the experiment but was markedly downregulated in the later phase. In Fig. 1B, western blot analysis revealed a pattern of SIRT1 protein expression levels similar to the PCR data. In addition, HIF-1a protein expression appeared to be upregulated in the first 12 h of the experiment, whereas it decreased in the later phase.

To investigate the role of SIRT1 in hypoxia-induced apoptosis in MC3T3-E1 cells, the protein levels of SIRT1 were altered. SIRT1 was overexpressed by using an adenovirus and knocked-down by si-RNA. The western blotting data demonstrated that the protein level of SIRT1 was successfully altered by knock-down or overexpression (Fig. 2A). The data demonstrated that SIRT1 overexpression in cells significantly increased cell viability and markedly suppressed the pace of apoptosis. The activity of the caspase 3/7 pathway was nullified by SIRT1 overexpression which interrupted the process of apoptosis response to hypoxia. By contrast, SIRT1 knockdown cells demonstrated the opposite phenotype (Fig. 2B-D). Taken together, these results suggested that SIRT1 served a protective effect in hypoxia-induced apoptosis in MC3T3-E1 cells.

To assess the mechanism of SIRT1 under hypoxia, a search was conducted for its partner proteins. Immunoprecipitation was performed to detect the co-expression of partner proteins. SIRT1 was immunoprecipitated from osteoblast lysates and partner proteins that were pulled down in the precipitate identified (Fig. 3). It was identified that Akt, a key protein in the apoptosis pathway, successfully interacted with SIRT1. The Akt protein pulled down with SIRT1 but not with the IgG negative control. To obtain further evidence for a SIRT1/Akt interaction, an inverse experiment was performed in which SIRT1 was intended to be pulled down by Akt. Lysine is normally an interaction residue for SIRT1, so it may also interact with Akt. To confirm this hypothesis, another immunoprecipitation was performed to demonstrate that Akt interacted with lysine; the resulting beads were analyzed by western blotting. These results demonstrated that Akt physically binds to SIRT1 and that lysine is capable of interacting with Akt.

During the apoptosis process, two signaling pathways are activated, the canonical and non-canonical. Previous studies have demonstrated that cytochrome c, apoptotic protease activating factor 1, and pro-caspase 9 released from the mitochondria interact with each other to form the apoptosome that drives the activation of caspase 3 (15,16). Due to the crucial role of caspases 3 and 9 in the apoptotic process, their protein expression was detected by western blotting. The hypoxia treatment potentiated apoptosis and the active forms of caspases 3 and 9 were increased. A high level of cleaved caspases 3 and 9 were detected. SIRT1 silencing treatment further increased the amount of cleaved caspases 3 or 9. In contrast, SIRT1 overexpression eliminated the expression of active forms of caspases under hypoxia (Fig. 4A). These findings suggest that SIRT1 may exert its regulatory effect on MC3T3-E1 cell apoptosis by regulating the ratio of pro-caspase 9/caspase 9 and pro-caspase 3/caspase 3.

The Bcl-2 family regulates the canonical pathway of apoptosis by activation or suppression. Of the Bcl-2 family members, Bax and Bcl-2 are recognized as two of the most important, that exert either pro- or anti-apoptotic effects in cells (17). In response to hypoxic stress, Bax and Bcl-2 protein levels were altered by the differing amounts of SIRT1 present in the MC3T3-E1 cells (Fig. 4A). Bax was abundant in SIRT1 silenced MC3T3-E1 cells. In contrast, the Bax level was significantly reduced in SIRT1 overexpressed cells. Bcl-2, the anti-apoptosis protein, demonstrated the opposite phenotype and was markedly increased in SIRT1 overexpression cells. Several proteins or transcription factors were additionally detected using western blotting or RT-qPCR (Fig. 4B). Levels of Akt, p65, TRAF2, and cIAP2 were significantly lower in SIRT1 silenced cells compared with SIRT1 overexpressed cells. Together, these results suggested that SIRT1 attenuated the apoptosis of MC3T3-E1 cells by regulating the expression levels of various proteins.