Cell culture and differentiation into osteoclasts

Mature osteoclasts were generated from bone marrow macrophages (BMMs) as described previously (Geng et al. 2017). Briefly, primary bone marrow cells were isolated from the femora of 5-week-old C57BL/6 mice. We cultured primary bone marrow cells in the complete medium containing 50 ng/mL recombinant M-CSF (R&D Systems, Minneapolis, MN, USA) for 3 days. Adherent cells at this stage were considered M-CSF–dependent BMMs and used as osteoclast precursors (cells at day 0). For osteoclastogenesis, the resultant BMMs were plated in 48-well plates and cultured in osteoclastogenic medium that consisted of 30 ng/mL M-CSF and 50 ng/mL RANKL for 4 days. We identified osteoclasts by staining for tartrate-resistant acid phosphatase (TRAP) activity. The cells were fixed with 4% paraformaldehyde and stained using the TRAP staining kit (Sigma-Aldrich, 387 A-1KT) based on the instructions received from the manufacturers. The TRAP+ multinucleated (> 3 nuclei) cells were counted as osteoclasts. According to the experimental requirements, 20IU/ml EPO (Sunshine Pharmaceutical, Shenyang, China), 0.5 mg/ml EMP9 (MedChemExpress, Wuhan, China), 20 µM TZD (Sigma, St. Louis, MO, USA), or 10 µM GW9662 (Sigma, St. Louis, MO, USA) were added into the osteoclast differentiation process.

Immunofluorescence staining

The cells were fixed in 4% formaldehyde for 15 min, then the membranes were broken with 0.1% Triton X-100 for 10 min. The cells were treated with a blocking buffer and then exposed to primary antibodies targetting EPOR or PPARγ at 4 °C overnight. Subsequently, the cells were incubated with secondary antibody-labeled fluorescent at room temperature for 1 h. The cell nuclei were stained with DAPI, and fluorescence images were acquired using confocal laser scanning microscopy (Smartproof 5, Carl Zeiss, Oberkochen, Germany).

Western blotting

The cells were washed twice with PBS and lysed in an ice-cold RIPA Lysis Buffer (Beyotime, China) containing protease inhibitors (Beyotime, China) for 20 min to extract total protein. Then, the supernatant was collected and centrifuged for 10 min at 12,000 g and 4 °C to obtain the protein sample. An equal amount of protein (50 µg) was mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Beyotime, China) and then heated at 95 °C for 5 min. Subsequently, the protein sample was separated using SDS-PAGE on 14% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then examined using specific primary antibodies against EPOR (ABclona; Catalog: A2917), phospho- (p-)Jak2, Jak2, p-ERK, ERK, GAPDH and PPARγ overnight at 4 °C, followed by incubation with corresponding secondary antibodies at room temperature for 1 h. Finally, immunoreactive bands were detected with ECL reagents. All antibodies were purchased from ABclona.

Actin ring formation assay

BMMs were seeded in confocal dishes and incubated in a complete inducing medium containing M-CSF and RANKL for four days to form actin rings. Subsequently, the cells were washed twice with PBS and fixed in 3.7% paraformaldehyde for 15 min. The cells were washed with PBS three times. Then, the cells were stained with rhodamine-conjugated phalloidin (Cytoskeleton, Inc., Denver, CO, USA), while the cell nucleus was stained with DAPI (Sigma–Aldrich) for 10 min. The cells were photographed using a confocal laser scanning microscopy.

Bone resorption assay

BMMs (2 × 104 cells/well) were cultured in Osteo Assay Surface 24-well plates (Corning, NY, USA) coated with a calcium phosphate substrate. BMMs incubated in complete inducing medium containing M-CSF and RANKL for 6 days. Subsequently, the cells were washed with 10% sodium hypochlorite and rinsed with water three times. The images were captured using an ordinary light microscope. The percentage of the resorbed bone surface area was quantified using ImageJ software.

RT-qPCR

The total RNA was extracted using TRIzol according to the instructions received from the manufacturer. Then, RT-qPCR was performed to evaluate the transcription of osteoclast-related genes, including Acp5, Ctsk, Mmp9, Calcr, NFATc1, and c-fos using a SYBR Green qPCR Master Mix Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s recommendations. The relative mRNA expression was normalized with GAPDH. The primers used in this experiment was as follows:

Acp5, forward 5′-CTGGAG TGCACGATGCCAGCGACA-3′ and reverse 5′-TCCGTGCTCGGCGATGGACCAGA-3′;

Ctsk forward 5′-GATACTGGACACCCACTGGGA-3′ and reverse 5′-CATTCTCAGACACAATCCAC-3′;

Mmp9, forward 5′- GGAGCACGGCAACGGAGAAG-3′ and reverse 5′- CCTGGTCATAGTTGGCTGTGGTG-3′;

Calcr, forward 5′-ATTTTGCCACTGCCTTTCAG-3′ and reverse 5′-ATTTTCTCTGGGTGCGCTAA-3′;

NFATc1, forward 5′-TGTTCTTCCTCCCGATGTCT-3′ and reverse 5′-CCCGTTGCTTCCAGAAAATA-3′;

c-fos, forward 5′-TTGCTGATGCTCTTGACTGG-3′ and reverse 5′-GGATTTGACTGGAGGTCTGC-3′;

GAPDH, forward 5′-AAATGGTGAAGGTCGGTGTG-3′ and reverse 5′-TGAAGGGGTCGTTGATGG-3′.

Animal treatments

The Animal Welfare Committee in Zhejiang University School of Medicine approved all animal care activities. Female C57BL/6 mice were randomly divided into four groups with 5 mice in each group at 10–12 weeks of age: (a) PBS, (b) rhEPO, (c) rhEPO + GW9962, and (d) rhEPO + ZOL. The rhEPO was administered intravenously at a dose of 5000 IU/kg three times per week for 2 months. The same volume of PBS, GW9962 (1 mg/kg), and ZOL (2 mg/kg,) was given to the control and rhEPO + ZOL group. rhEPO was obtained from Sunshine Pharmaceutical (Shenyang, China) for patient care.

Micro-computed tomography analysis

After mice were sacrificed, the femora (1 per mouse) were soaked in ethanol. We used a micro-CT (Analyze 12.0, PerkinElmer) to scan the femurs. We scanned the entire length of the femur at a pixel size of 15 μm, and analyzed the results according to the manufacturer’s instructions. Region-of-interest (ROI) was defined from 0.225 mm (15 image slices) to 2.475 mm (165 image slices), with the growth plate slice defined as 0 mm. We performed an analysis of bone-related parameters in the ROI region. We used 3D models to enhance the interpretation of the images by reconstructing the data specifically from the ROI region. The software (Analyze 12.0, PerkinElmer) was used to analyze trabecular microstructural parameters, including bone mineral density (BMD), trabecular number (Tb. N), trabecular bone volume per tissue volume (BV/TV), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp).

TRAP staining of tibias

After animal sacrifice, the tibias per mouse were fixed in 4% paraformaldehyde for 48 h. After the samples were decalcified in 0.5 M EDTA for 4 weeks, they were embedded in paraffin. Five-micrometers-thick sections of the tibias were performed the tartrate-resistant acid phosphatase (TRAP) staining using the standard protocol.

ELISA Analysis and blood analysis

After the final dose of injection, blood was collected by enucleation of the eyeballs, and serum was collected by separation from the clotted blood for ELISA assay and blood analysis. 0.5 mL mouse whole blood was collected in a 2 mL centrifuge tube containing 2.5 µL of 2% heparin sodium. Then, blood parameters (hematocrit, RBCs and hemoglobin) were measured using a Sysmex automated blood cell counter (Sysmex XE-2100 and XE-5000). 1mL mouse whole blood was collected in a 2 mL centrifuge tube. Serum was collected from the supernatant of centrifuged mouse whole blood. Then, bone metabolism level was evaluated by measuring the concentration of serum C-terminal telopeptides of type I collagen (CTX-1). Serum CTX-1 levels were analyzed using a mouse CTX-1 EIA kit (Immunodiagnostic Systems) according to the manufacturer’s instructions.

Statistical analysis

The data is presented as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 20.0 (SPSS Science Inc., Chicago, Illinois). Statistical significance was assessed using a one-way analysis of variance (ANOVA) analysis. A significant difference was considered as a P value < 0.05.