Animals
Male and female Balb/c mice, aged 4–6 weeks, were provided by the Experimental Animal Center of The Third Military Medical University and housed in a specific pathogen-free animal facility of Laboratory Animal Centre in the Second Affiliated Hospital of Army Medical University. All experiments in this study were approved by the Animal Experiment Ethics Committee of Army Medical University institute
Cell culture
Balb/c mice BM-MSCs, complete medium containing and osteogenesis inducing medium were purchased from Cyagen Biosciences Inc. Guangzhou, China. Cells were cultured at 37 °C under 5% CO2 atmosphere. The culture medium was replaced every 2 to 3 days. Cells at passage 11 (P8) were used in subsequent experiments.
Pre-conditioning irradiation
For all irradiation procedures, both cells and animals were total body irradiated (TBI) with a single dose of 60Co-γ using an irradiation source[3] with a source-surface distance of 150 cm and at a rate of 0.69 Gy/min at the Irradiation Research Centre of the Third Military Medical University. The dose for cells was 9 Gy and for animals was 5 Gy.
Reagents
The mouse recombinant IL-12 was purchased from Biolegend, San Diego, CA, USA. Small interfering RNA (siRNA) targeting mouse Tyk2 was chemically synthetic in Ribo Bio Co. Ltd., Guangzhou, China. Small interfering RNA targeting mouse IL-12Rβ1 were purchased from Santa Cruz Biotechnology Co. The inhibitor of STAT3, cryptotanshinone (CPT), was purchased from Med Chem Express. The bone marrow wash medium, StemSpan™ SFEM culture medium, the methylcellulose-based medium and the culture plates, SmartDish™ plate were purchased from Stemcell Technologies, Canada.
The primary antibodies used for western blotting were following: anti-IL-12Rβ1 (Bioss), anti-IL-12Rβ2 (Bioss), anti-RUNX2 (Runt-related transcription factor 2, Santa Cruz Biotechnology), anti-Osteoglycin (OGN, Santa Cruz Biotechnology), anti-β-actin (Santa Cruz Biotechnology), anti-phosphorylated STAT3 (Tyr705) mouse monoclonal (mAb) (Cell Signaling Technology), anti-STAT3 (124H6) mouse mAb (Cell Signaling Technology), rabbit anti-TYK2 antibody (Abnova, Taipei, Taiwan).
Quantitative polymerase chain reaction
Total RNA was isolated using Trizol reagent (Invitrogen. China) and the first-strand cDNA was synthesized using a reverse transcription reagent kit (TOYOBO, FSK-100, Osaka, Japan) accordance with the manufacturer’s instructions. Next, real-time qPCR (RT-qPCR) was performed in triplicate using Fast Start Universal SYBR Green Master Mix (Roche, Germany). The data were analysed using the delta-delta Ct method. Experiments were performed in triplicate.
The sequences of the primers used in RT-qPCR are as follows: GAPDH forward, 5′-GACATCAAGAAGGTGGTGAAGC-3′; GAPDH reverse, 5′-GAAGCTGGAAGAGTGGGAGTT-3′; Runx2 forward, 5′-GTGCCCAGGCGTATTTCAGATG-3′; Runx2 reverse, 5′-GCGGGGTGTAGGTAAAGGTCGC-3′; Ogn forward, 5′-GTGGTCACATGGATAGCCTTTAGTC-3′; Ogn reverse, 5′-GAGCATATTTAGTTTGTTTGGGTGA-3′; IL-12Rβ1 forward, 5'ATGCGCTGGTGGTCGAGATGC-3'; IL-12Rβ1 reverse, 5'CCCGGCTCCGCAGTCTTATG-3'; IL-12Rβ2 forward, 5'CGACGCTCTCAAAACTCACATCC-3'; IL-12Rβ2 reverse, 5'TTTGCCGGAAGTAACGAATTGAG-3'; Tyk2 forward, 5'-GTGCCTTCCGTGTTCAGCGTGTG-3'; Tyk2 reverse 5'-GCCCAGAACGAATAGACTCAGGAA-3'.
Western blotting
Total protein lysates were extracted with cell lysis buffer (Beyotime, Shanghai, China) and were denatured by boiling. Next, the cell lysates were separated on 4–12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (PVDF Western Blotting Membranes, Roche). The membranes were blocked in PBS buffer (50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween [ pH 7.6 ]) supplemented with 5% non-fat dry milk and were incubated with the appropriate antibodies for 12 hours. An HRP-labelled secondary antibody and a chemiluminescent detection system (Phototope-HRP western blot detection kit; New England Biolabs, Ipswich, MA) were used for developing the blots.
Hematopoiesis and osteobalsts detections
The peripheral blood cell counts (PBCC) were estimatied by analyzing the eyeball blood with the automatic animal blood cell analyzer (Prandre XFA6030, Nanjing Prande Medical Equipment Co., Ltd., China). For the hematopoiesis of bone marrow, the bilatteral femur were removed and fixed in 10% formalin and embedded in paraffin wax for micro-sectioning at 5 µm and routine hematoxylin & eosin (HE) staining. All slides were examined under microscope (Axio Imager 2, Zeiss, Germany) and the volume of bone marrow hematopoietic tissues were detected by image analysis and reporting system (YC.YX-2050, Chongqing telemedicine equipment Co., Ltd. China.).
For bone marrow HSPCs colony assays, mice were euthanized by injecting 150 mg/kg sodium pentobarbital and their bilatteral femur were removed. The bone marrows were aseptically flushed into serum free expansion medium (StemSpan™ SFEM, StemCell Technologies Inc, Vancouver, BC, Canada) using a syringe fitted with a 2 l-gauge needle. Cells were counted and cultured in methylcellulose media (MethoCult GF, StemCell Technologies Inc, Vancouver, BC, Canada) following the manufacturer’s instruction. A total of 300000 cells from unirradiated mice were used as a normal control (Normal). The colony-forming units (CFU) of granulocyte (CFU-G) and macrophage (CFU-M), CFU-granulocyte macrophage (CFU-GM), CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) were analyzed according to the technical manual, mouse CFU assays using methoCult™(Stemcell Technologies, Canada) in situ by light microscope (Leica DMIRB, Germany).
Osteogenic differentiation and bone formation detection of BM-MSCs
BM-MSCs committed osteogenic differentiation detection were analyzed by mineralized matrix staining for calcium mineral deposits using alizarin red S staining kit (Cyagen, China). ALP activities was tested using ALP staining complied with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue-tetrazolium (BCIP/NBT) alkaline phosphatise colour development kit (Beyotime Biotechnology, Shanghai, China). The mineralized matrix were examined and photographed using a light microscope (Leica DMIRB, Germany). The bone mineral densities were analyzed at the bottom of the distal growth plate, where the epiphyseal cap structure completely disappeared and continued for 95 slices (10.5 µm/slice, using Scanco Viva CT40) towards the proximal end of the femur.
Cells transplantation assays
Before transplantations, irradiated mice were anesthetized with 20 µg/kg pentobarbital sodium, using a 10 µl microliter syringe purchased from Gaoge indsstrial and trading co. LTD., and subsequently cells suspended in a minimal volume of 3∝ l PBS and 10000 cells were injected intrafemorally through the patellar surface. The whole cells transplantaions were carried out within 1 hour after TBI.
Transfection, inhibition and immunofluorescence assays
Briefly, cells transfected with special siRNA (final concentration, 50 nM) by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. After 72 hours of transfection, cells were co-cultured with or without 0.2 ng/ml IL-12 for 24 hours. For the inhibition of STAT3, the inhibitor were added in the culture medium at 50 nM for co-culturing 24 hours, and then cells were co-cultured with or without 0.2 ng/ml IL-12 for 24 hours. And then all the cells samples were collected for further detections.
Cells for immunofluorescence test were fixed with 4% formaldehyde and then were incubated overnight at 4 °C with a primary antibody and were incubated with FITC-labelled secondary antibody (Zhongshan Golden Bridge, China) for 1 h at RT. After each step, the prepared specimens were counterstained with 5 µg/ml 4,6-diamidino-2-phenylindole and were observed with a laser scanning confocal microscopy (LSCM) (Olympus, Japan).
Statistical analysis
All data were presented as the mean ± SEM. Statistical analysis was performed using GraphPad Prism version 6.0. An independent unpaired t test was used to compare data obtained from the experimental groups with those obtained from the control group. The results were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.