Cell culture
MC3T3-E1 cells (Cell bank of the Chinese Academy of Sciences, Shanghai, China) 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) and penicillin‑streptomycin solution (100 U/mL penicillin; 100 μg/mL streptomycin; Gibco; Thermo Fisher Scientific), in a humidified atmosphere with 5% CO2 at 37°C.
Cell viability assay
Cell viability assessment was performed using a Cell Counting Kit-8 (CCK-8, Signalway Antibody, MD, USA). MC3T3-E1 cells (1×104/well) were seeded in 96-well plates, incubated overnight and then treated with rapamycin at various concentrations 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 nM for 6, 12, 24, 36 and 48 hours. Cell viability was determined by CCK-8. Ten microliters of CCK-8 reagent were added to each well and incubated at 37°C for 3 h, until the media turned yellow. Absorbance was measured at 450 nm in a spectrophotometer.
Analysis of the cell cycle
A Cell Cycle Detection kit (KeyGen Biotech Co. Ltd, Nanjing, China) was used to assess the cell cycle. Following treatment with 50 nM rapamycin for 48 hours, 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.
Western blot analysis
The protein level were determined by Western blotting. MC3T3-E1 osteoblasts were seeded into 100 mm dishes and cultured in complete α-MEM to 70% confluency, following which the cells were treated with 50 nM rapamycin for 6, 12, 24, 36 and 48 hours. Total proteins were extracted following lysis of the cells in RIPA buffer at 4°C for 1 h. The lysates were centrifuged at 20000 rpm for 30 min, and the protein concentration was determined using a BCA protein quantification kit. Equal amounts of protein (30 μg) were loaded into each well of a 7.5–15% SDS-PAGE gel and transferred to PVDF membranes (Millipore Corp., Bedford, MA, USA). Membranes were incubated overnight at 4°C with the primary antibodies p62 (1:1000), LC3-II (1:1000), mTOR (1:1000), p-mTOR (1:1000), 4E-BP1 (1:1000), p-4E-BP1 (1:1000), S6K1 (1:1000), p-S6K1 (1:1000), Akt (1:1000), p-Akt (1:2000), phosphatidylinositol 3-kinase (PI3K) (1:1000), runt-related transcription factor 2 (Runx2) (1:1000), GAPDH (1:3000) (Cell Signaling Tehnology, Beverly, MA, USA), osterix (1:1000), and alkaline phosphatase (ALP) (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Adding secondary antibody (goat anti-rabbit IgG-HRP, 1:5000, Santa Cruz) incubated for 2 h. The specific bands were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific) and imaged with an Alpha Imager HP (ProteinSimple, San Jose, CA, USA). The band density was quantified using the ImageJ image processing program (National Institutes of Health, Bethesda, MD, USA. software version 1.5b).
Immunofluorescence staining
MC3T3-E1 osteoblasts were seeded in 24-well plates with culture slides (Beyotime Institute of Biotechnology, Shanghai, China) and upon reaching 50% confluency, were treated with 50 nM rapamycin for 12, 24, 36 and 48 hours. The cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and permeabilized with 0.5% Triton X-100 for 2 min at room temperature. After blocking for 2 h with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature, the cells were incubated overnight with anti-LC3-II antibody (1:200) at 4°C. After 1 h incubation with Fluorescein (FITC)-conjugated secondary antibody (1:5000) (Santa Cruz), the cells were counterstained with DAPI for 10 min at room temperature. The stained cells were imaged using confocal fluorescence microscopy (magnification, ×200, DS-U3 Nikon Eclipse CI; Nikon Corporation, Tokyo, Japan).
Reverse transcription and real-time PCR
MC3T3-E1 osteoblasts were seeded at a density of 5×104 cells/well in 100 mm dishes for 3 days until the cells reached 70% confluence. The cells were treated with 50 nM rapamycin for 6, 12, 24, 36 and 48 hours as described, and total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific), according to the manufacturer's protocol. Total RNA was extracted and quantified by scanning spectrophotomer. A total of 1 μg of RNA was used in the reverse transcription reaction using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's protocols. For gene-specific primed cDNA synthesis, the reaction was conducted for 60 min at 42°C. Then, for random hexamer primed synthesis, incubate for 5 min at 25°C. RT was terminated by heating at 70°C for 5 min. Real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific) on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems 7500 System Sequence Detection System, software version 2.6.2; Thermo Fisher Scientific, Inc.). The PCR conditions were as follows: 95°C preheating for 1 min, followed by 40 cycles of 95°C for 15 s (denaturation), 60°C for 20 s (annealing), and 72°C for 32 s (elongation). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the inner reference. The threshold cycle (Ct) values were calculated using the StepOnePlus system software (Applied Biosystems). The relative expression of each mRNA was calculated by the 2−(ΔCt) method. The primer sequences were as follows: LC3-II forward 5'-CCGACCGCTGTAAGGAGG-3'; LC3-II reverse 5'-AAGCCGAAGGTTTCTTGGGA-3'; p62 forward 5'-GTGTGCCCAGACTACGACCT-3'; p62 reverse 5'-GACTCAGCTGTAGGGCAAGG-3; mTOR forward 5'-CGCTACTGTGTCTTGGCATC-3'; mTOR reverse 5'-GGTTCATGCTGCTTAGTCGG-3'; ALP forward 5'-TGCCCTGAAACTCCAAAAGC-3'; ALP reverse 5'-CTTCACGCCACACAAGTAGG-3; Runx2 forward 5'-CCCAGCCACCTTTACCTACA-3'; Runx2 reverse 5'-TATGGAGTGCTGCTGGTCTG-3; osterix forward 5'-CATCCCTATGGCTCGTGGTA-3'; osterix reverse 5'-TGGGTTAAGGGGAGCAAAGT-3'; GAPDH forward 5'-ATGGGTGTGAACCACGAGA-3'; GAPDH reverse 5'-CAGGGATGATGTTCTGGGCA-3'.
Statistical analysis
All statistical tests were performed by SPSS20.0 statistics software (SPSS, Chicago, USA). All experiments were performed three times. Values are expressed as the mean ± standard deviation (SD). Differences between two groups were analyzed by the LSD and Duncan’s test. Independent samples t test or one-way analysis of variance was used in comparison between groups. All statistical analysis was performed using SPSS for Windows ver. 19.0 (IBM Corp., Armonk, NY, USA). p<0.05 was considered to indicate a statistically significant difference.