Cell culture
Human cementoblast cells (HCEM2) were a gift of Hiroshima University, Japan. HCEM2 cells were cultured in Dulbecoo’s Modified Eagle Medium (DMEM) (Wako, Tokyo, Japan) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin under 5% CO2 at 37°C in a humidified environment. The cells were sub-cultured at 70-80% confluency. About 1.2 x 105 HCEM2 cells were seeded in 6-well plates with 2 ml normal growth medium and 24 h later were treated with 5 mM NaF for 5 min.
Animals
C57BL/6 mice (10-week-old, n=12, male, body weight 29.4 ± 0.8 g) were obtained from Japan SLC (Shizuoka, Japan) and were individually housed under a 12:12 h light-dark cycle in pathogen-free conditions. The mice were divided into 2 groups: a control group (n=6) and a 5 mM NaF treated group (n=6). The mice were provided water with or without 5 mM NaF at the age of 12 weeks and stopped at 58 weeks. Mice were sacrificed by cervical dislocation under deep anesthesia with a mixture of medetomidine hydrochloride at a dose of 0.3 mg/kg, midazolam at a dose of 4 mg/kg and butorphanol tartrate at a dose of 5 mg/kg (0.1 ml/10g body weight). All animal experiments were performed after approval by the Institute’s Ethics Committee at Nihon University School of Dentistry at Matsudo (AP17MD015).
Measurement of alveolar bone resorption and micro-computed tomography
The distance between the cemento-enamel junction and the alveolar bone crest at seven buccal sites per mouse was measured, standardized to provide measurements in millimeters. Three-dimensional (3D) changes for each mandibular bone were captured using micro-computed tomography (micro-CT) (R_mCT2, Rigaku Corp., Tokyo, Japan) under the following exposure conditions: tube voltage, 90 kV; tube current, 200 μA; voxel size, 20 × 20× 20 μm.
MTS assay
The MTS assay was performed as previously described [29]. Briefly, 3 x 103 HCEM2 cells per well were seeded and cultured in 96-well plates for 24 h. Serum-free medium was changed after 24 h and then replaced with 5 mM NaF for 5 min. Cell viability was assessed by the MTS assay after NaF supplementation according to the manufacturer’s instructions. Cell Titer 96® Aqueous One Solution Reagent (Promega, Madison, WI, USA) was used to detect cell viability. The results were quantitated by absorbance at 490 nm.
Flow cytometry
Cell apoptosis was detected using a BD Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, CA, USA) performed as per the manufacturer’s instructions. HCEM2 cells were trypsinized and collected after treatment with 5 mM NaF for 5 min. One x 106 cells/mL were resuspended in fresh culture medium and stained with or without Annexin V-FITC and Propidium Iodide for 15 min. The cells were then washed with binding buffer and immediately analyzed using flow cytometry (FACS Calibur, BD Bioscience, San Jose, CA, USA).
Western blot
HCEM2 cells were solubilized by RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Twenty µg protein were applied on each polyacrylamide-SDS gel (Wako, Osaka, Japan) and then transferred to PVDF membranes (Millipore, St. Louis, MO, USA). After blocking with 5% skim milk, the membranes were incubated with anti-ATG5 (1:500, ab108327, Abcam, Tokyo, Japan), anti-Beclin1 (1:500, ab62557, Abcam, Tokyo, Japan) anti-LC3-I/II (1:1000, #12741, Cell Signaling Technology, Danvers, MA, USA), anti-p62 (1:1000, ab56416, Abcam, Tokyo, Japan), anti-Bax (1:1000, ab32503, Abcam, Tokyo, Japan), anti-p-NF-κB (1:800, bs-3543R, Bioss, Woburn, MA, USA), anti-NF-κB (1:800, #4746, Cell Signaling Technology, Danvers, MA, USA), anti-SOD1 (1:500, ab13498, Abcam, Tokyo, Japan), anti-HIF1-α (1:500, ab2185, Abcam, Tokyo, Japan), anti-Cleaved-caspase3 (1:500, ab49822, Abcam, Tokyo, Japan) or anti-GAPDH (1:1000, #2118, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:2000; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. An ECL Plus Western Blotting Detection System (GE Healthcare, Tokyo, Japan) was used to visualize the images of western blots.
RT-PCR
RNA was extracted from HCEM2 cells using a RNeasy Mini Kit (Qiagen KK, Tokyo, Japan) according to the manufacturer’s protocol. Briefly, cDNAs were transcribed from 1 µg of each RNA using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed to detect expression of the following target genes using TaqMan gene Expression Assays: ATG5 (Assay ID Hs00169468_m1), Beclin1 (Assay ID Hs01007018_m1), LC3-I (Assay ID Hs01076567_g1), LC3-II (Assay ID Hs00797944_s1) and ACTB (Assay ID Hs01060665_g1). Each detection was performed in triplicate.
Immunohistochemical staining
Tissues of the upper jaw were fixed with 4% paraformaldehyde (Wako, Osaka, Japan) and decalcified with 10% EDTA (pH: 8.0). The specimens were subjected to antigen retrieval (pH: 6.0) and peroxidase blocking and were then incubated with anti-ATG5 (1:75, ab108327, Abcam, Tokyo, Japan), anti-Beclin1 (1:100, ab62557, Abcam, Tokyo, Japan), anti-Cathepsin K (1:50, LS-B2512, Abcam, Tokyo, Japan), anti-RANKL (1:50, NB100-80849,Novus, Centennial, CO, USA), anti-Bax (1:100, ab32503,Abcam, Tokyo, Japan), anti-p-NFkB (1:100, bs-3543R, Bioss, Woburn, MA, USA), anti-HIF1-a (1:100, ab2185, Abcam, Tokyo, Japan) or anti-8OHdG (1:20, MOG-020P, JaICA, Shizuoka, Japan) overnight at 4°C. Further, the slides were then incubated with secondary antibodies (Rat MAX-PO, Nichirei Bioscience, Tokyo, Japan) at room temperature for 30 min. Images were captured using a microscope (OLYMPUS, Tokyo, Japan).
Immunofluorescent staining
As described above, the slides were incubated with anti-Runx2 (1:50, 5356-1, Epitomics, Tokyo, Japan), anti-Osterix (1:75, ab94744, Abcam, Tokyo, Japan), anti-ATG5 (1:75, ab108327, Abcam, Tokyo, Japan) or anti-Beclin1 (1:100, ab62557, Abcam, Tokyo, Japan) overnight after antigen retrieval and peroxidase blocking. The slides were incubated with anti-rat IgG-Alexa Fluor 488 secondary antibody at room temperature for a duration of 60 min in a dark chamber. A fluorescence microscope was used to capture images (OLYMPUS, Tokyo, Japan).
Statistical analysis
Statistical analysis was performed by SPSS 16.0 and the results were assessed by an independent two-tailed Student’s t-test or analysis of variance (ANOVA). p value less than 0.05 showed significant difference.