MC3T3-E1 cells (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (high glucose; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin-streptomycin solution (100 U/ml penicillin; 100 μg/ml streptomycin; Gibco; Thermo Fisher Scientific, Inc.), in a humidified atmosphere with 5% CO₂ at 37°C.

Cell viability assessment was performed using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). MC3T3-E1 cells (1×10⁴/well) were seeded in 96-well plates, incubated overnight and then treated with DEX at various concentrations 0.1, 1 and 10 μM for 24 h. The absorbance values at 450 nm were measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Furthermore, in order to assess the connection of DEX-induced autophagy with apoptosis, 3-methyladenine (3-MA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used as a common anti-autophagy factor. Cells were divided into four groups, including normal control, 1 μM DEX, 5 mM 3-MA (cells cultured with 5 mM 3-MA for 24 h) and 5 mM 3-MA + 1 μM DEX group (cells cultured with 5 mM 3-MA + 1 μM DEX for 24 h). In addition, in order to investigate the association between ROS induced by DEX and cell apoptosis, 10 mM N-acetylcysteine (NAC; Beyotime Institute of Biotechnology, Haimen, China) was added to MC3T3-E1 cells treated with DEX. NAC is a commonly used antioxidant. Cells were divided into normal group, 1 μM DEX group, 10 mM NAC group (cells cultured with 10 mM NAC for 24 h) and 10 mM NAC + 1 μM DEX group (cells cultured with 10 mM NAC + 1 μM DEX for 24 h).

An Annexin V-fluorescein isothiocyanate (FITC) apoptosis Detection kit (KeyGen Biotech Co., Ltd., Nanjing, Jiangsu, China) was used to determine the level of cellular apoptosis. MC3T3-E1 cells (4×10⁶/well) were cultured in 6-well plates overnight and then treated as described above. In brief, cells were suspended in 500 μl 1X binding buffer and incubated in the dark with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) for 10 min at room temperature. A flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze cellular apoptosis.

A Cell Cycle Detection kit (KeyGen Biotech Co., Ltd.) was used to assess the cell cycle. Following treatment with DEX, the MC3T3-E1 cells were trypsinized, collected and washed with PBS. Subsequently, 70% cold ethanol was added to fix the cells for 2 h at room temperature or overnight at 4°C, and PBS was used to wash away the fixing solution. Cells were incubated with 0.4 ml PI containing 0.1 ml RNase A at 37°C for 30 min. Finally, cell cycle distribution was analyzed by measuring the DNA content using a flow cytometer.

Autophagy is characterized by the formation of acidic vesicles, as described previously, and MDC is often used as a selective fluorescent marker for autophagic vacuoles in vivo (15). MC3T3-E1 cells (4×10⁶/well) were seeded onto sterile coverslips placed in a 6-well plate and allowed to attach for 24 h. As aforementioned, 0.05 mmol/l MDC in PBS was added to label the autophagic vacuoles for 10 min at 37°C, and cells were washed three times with PBS. MC3T3-E1 cells were observed under a fluorescence microscope (Olympus BX53; Olympus Corp., Tokyo, Japan). Excitation wavelength was 335 nm, and emission wavelength was 512 nm. Average fluorescence intensity analysis was performed using Image-Pro Plus v6.0 software (Media Cybernetics Inc., Rockville, MD, USA).

A ROS Assay kit (KeyGen Biotech Co., Ltd.) was used for active ROS detection using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which is a type of ROS-sensitive dye (16). The DCFH-DA reagent must be diluted to 10 μmol/l in serum-free medium prior to use. Following the experimental treatments, 10 μmol/l DCFH-DA was added to cells with subsequent incubation for 30 min at 37°C in a humidified incubator, and cells were washed three times with serum-free medium and collected. A flow cytometer was used to quantify the relative fluorescence intensities.

Total protein was extracted from treated MC3T3-E1 cells with radioimmunoprecipitation assay lysis buffer (Tris 50 mM, NaCl 150 mM, Nonidet P-40 1%, sodium deoxycholate 0.5% and SDS 0.1%; Beyotime Institute of Biotechnology, Haimen, China) containing 1% phenylmethylsulfonyl fluoride (100 mM; Beyotime Institute of Biotechnology). The lysates were centrifuged at 12,000 × g for 15 min at 4°C. Supernatants were collected and the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) was used to detect the protein concentration. Equivalent amounts (25 μg) of protein were separated by 12% SDS-PAGE and subsequently transferred onto a polyvinylidene difluoride membrane (Merck KGaA). Membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (0.05%) at room temperature for 1 h and incubated overnight with primary antibodies. Following incubation with the corresponding secondary antibody, horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (1:10,000 dilution; cat. no. 7074P2; Cell Signaling Technology, Inc., Danvers, MA, USA), at room temperature for 2 h, membranes were washed three times with PBS. Finally, according to the manufacture's protocols, the blotting proteins were visualized with ECL plus reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Antibodies used for the western blot analysis in the present study were as follows: Rabbit anti-activating transcription factor 4 (ATF4; 1:1,000 dilution; cat. no. 11815), rabbit anti-CCAAT/enhancer-binding protein homologous protein (CHOP; 1:1,000 dilution; cat. no. 2895), rabbit anti-poly[ADP ribose] polymerase (total PARP; 1:2,000 dilution; cat. no. 9542), rabbit anti-caspase-3 (total caspase-3; 600 dilution; cat. no. 96621) were obtained from Cell Signaling Technology, Inc. In addition, rabbit anti-Beclin1 (1:1,000 dilution, cat. no. ab62557), rabbit anti-LC3B (1:1,000 dilution, cat. no. ab51520), rabbit anti-P62 (1:1,000 dilution; cat. no. ab91526) and rabbit anti-cyclin-dependent kinase 2 (CDK2; 1:1,000 dilution; cat. no. ab32147) were obtained from Abcam (Cambridge, UK). Additionally, rabbit anti-GAPDH (1:1,000 dilution; cat. no. AB-P-R 001; Hangzhou Goodhere Biotechnology Co., Ltd., Hangzhou, China) was used as loading control. ImageJ v1.47 software (National Institutes of Health, Bethesda, MD, USA) was used to perform relative intensity of each protein band analysis.

All experiments were performed three times. Statistical analysis was performed with SPSS 19.0 statistical software (IBM Corp., Armonk, NY, USA). One-way analysis of variance followed by Tukey's post hoc test was used for multiple comparisons, and Student's t-test for comparisons between two groups. Values are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The effect of DEX on cell viability was evaluated with a CCK-8 assay. As illustrated in Fig. 1A, the amount of viable MC3T3-E1 cells was significantly inhibited by DEX in a dose-dependent manner (all P<0.01), and at 10 μM DEX, the cell viability reached 70% of that of the control group. In addition, the cellular apoptosis assay demonstrated that compared with that in the control group (2.73%), the apoptotic rates in the DEX-treated groups were 7.9, 17.2 and 21.07% at DEX concentrations of 0.1, 1 and 10 μM, respectively (P<0.05, P<0.01 and P<0.01, respectively; Fig. 1B and C).

Furthermore, western blotting was performed to evaluate the expression of PARP and caspase-3, which are early markers of apoptosis. As presented in Fig. 1D and E, DEX increased the activation of caspase-3 and PARP, which further confirmed that DEX promoted apoptosis of MC3T3-E1 cells. These results demonstrated that DEX not only reduces cell viability, but also promotes apoptosis. In addition, CHOP and ATF4 are key factors of ER stress. The expression of CHOP and ATF4 was gradually enhanced with increasing concentration of DEX (Fig. 1D and F). These results indicated that DEX may promote apoptosis by activating ER stress.

The results of the cell cycle analysis are presented in Fig. 2A and B. DEX at concentrations of 1 and 10 μM induced a significant increase in the number of cells in G0/G1 phase, but decreased the number of cells in S phase, indicating that DEX inhibited cell cycle progression (both P<0.05). Subsequently, the expression of CDK2, which is essential for the G1/S transition, was detected. As illustrated in Fig. 2C and D, the expression of CDK2 was inhibited in MC3T3-E1 cells after treatment with DEX at concentrations of 1 and 10 μM (P<0.05, P<0.01). These results suggested that DEX may induce G0/G1 arrest in MC3T3-E1 cells due to decreased CDK2 protein expression.

To investigate the effect of DEX on autophagy in MC3T3-E1 cells, MDC staining and western blotting were performed. After treatment with DEX for 24 h, fluorescence intensity was analyzed (Fig. 3A and B). The average fluorescence intensities in the 0.1, 1 and 10 μM DEX-treated groups were increased to 0.18, 0.22 and 0.25, respectively, which were significantly different from the average fluorescence intensity of the control group, 0.11 (P<0.05, P<0.01 and P<0.01, respectively). Furthermore, in DEX (1 μM) and DEX (10 μM) groups, the expression levels of autophagy markers, including LC3B-II, Beclin1 and P62, were gradually increased, but the levels of P62 were reduced, indicating the stimulatory role of DEX on autophagic flux (P<0.05, P<0.01; Fig. 3C and D, respectively). These results indicated that DEX may promote the autophagy of MC3T3-E1 cells.

3-MA inhibits autophagy by blocking autophagosome formation via the inhibition of class III phosphoinositide-3 kinase (17). To clarify the possible association between autophagy and apoptosis after treatment with DEX, the effect of 3-MA on viability and apoptosis was investigated. MC3T3-E1 cells were treated with DEX (1 μM), 3-MA (5 mM) or 3-MA (5 mM) + DEX (1 μM). As demonstrated in Fig. 4A, the viability of MC3T3-E1 cells decreased to 76.5% in the presence of DEX (1 μM); however, after treatment with 3-MA (5 mM) + DEX (1 μM), the viability was significantly increased to 90.1% (P<0.001). Furthermore, as presented in Fig. 4B and C, the apoptotic rate of MC3T3-E1 cells treated with DEX (1 μM) was 24.3%, while it significantly decreased to 18.56% in the 3-MA (5 mM) + DEX (1 μM) group (P<0.05). Inconsistencies in the apoptotic proportion in the DEX (1 μM) group between Figs. 1C and Fig. 4C may be due to technical issues, including different batches of kit. These results indicated that 3-MA reversed the DEX-induced inhibition of viability and promotion of apoptosis in MC3T3-E1 cells.

Furthermore, the expression levels of apoptosis- and autophagy-associated proteins were detected (Fig. 4D). The expression levels of the autophagy-associated proteins LC3B-II and Beclin1 were increased in the DEX (1 μM) group, which was inhibited by simultaneous treatment with 3-MA (5 mM; P<0.05; Fig. 4E). In addition, it was demonstrated that the activation/expression of the apoptosis-associated proteins PARP and caspase-3 was weakened after treatment with 3-MA (5 mM) + DEX (1 μM), compared with that in the DEX (1 μM) group (P<0.05; Fig. 4F). These results suggested that DEX may promote MC3T3-E1 cell apoptosis by inducing autophagy.

To examine how DEX affects the production of ROS in MC3T3-E1 cells, flow cytometry was performed to quantify the fluorescence intensity. In this experiment, NAC was used as an antioxidant. As illustrated in Fig. 5A and B, the mean ROS-associated fluorescence intensity in the DEX (1 μM) group was increased compared with that in the control group, which was significantly inhibited by simultaneous treatment with NAC (10 mM; P<0.01). Furthermore, the cell viability in the NAC (10 mM) + DEX (1 μM) group was 89.3% of that in the control group, which was significantly higher than that in the DEX (1 μM) group (77.8% of control; P<0.01; Fig. 5C). These results demonstrate that NAC inhibited the effect of DEX on MC3T3-E1 cell proliferation following co-treatment. Furthermore, cellular apoptosis was detected (Fig. 5D and E), revealing that the apoptotic rate of MC3T3-E1 cells treated with NAC (10 mM) + DEX (1 μM) was 17.7%, which was significantly decreased compared with that in the DEX (1 μM) group (25.97%; P<0.01). These results indicated that activation of intracellular ROS was involved in the DEX-induced inhibition of viability and the promotion of apoptosis in MC3T3-E1 cells.

Subsequently, the expression of autophagy- and ER stress-associated proteins was quantified by western blot analysis (Fig. 5F). As illustrated in Fig. 5G, the protein levels of LC3B-II and Beclin1 were decreased, but P62 expression was increased in the NAC (10 mM) + DEX (1 μM) group when compared with that in the DEX (1 μM) group (P<0.05). This result indicated that DEX activated autophagy via inducing ROS-associated stress. In addition, it was demonstrated that the expression of ATF4 and CHOP was also inhibited by NAC, indicating that ROS-associated stress also elevated ER stress (P<0.05; Fig. 5H). Therefore, the effect of DEX on cell viability, ROS stress, apoptosis and protein expression was attenuated by NAC. These results demonstrated that DEX promoted the production of ROS, which enhanced apoptosis through the induction of autophagy and ER stress in MC3T3-E1 cells.