Preparation of rhBMP-2
C-rhBMP-2 was purchased from National Institute for Biological Standards and Control (Hertfordshire, UK). The C-rhBMP-2 was prepared by reconstitution with 1 mL of distilled water in accordance with the manufacturer’s protocol. E-rhBMP-2, a homo-dimeric protein with a mature rhBMP-2 sequence, was provided by Daewoong Pharmaceutical Co., Ltd. (Seoul, Republic of Korea). The E-rhBMP-2 was prepared in a buffer containing 25 mg/mL glycine, 3.7 mg/mL glutamic acid, 5 mg/mL sucrose, 0.1 mg/mL NaCl, and 0.1 mg/mL polysorbate 80.
Surface plasmon resonance analysis
Surface plasmon resonance (SPR) measurements were performed using a Biacore T200 instrument (GE Healthcare, Pittsburgh, PA, USA). BMP receptor type 1A (BMPR1A), BMP receptor type 1B (BMPR1B), and BMP receptor type 2 (BMPR2) were purchased from Thermo Fisher Scientific Co. (Rockford, IL, USA). A mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.1 M N-hydroxysuccinimide was injected over both the sample (E-rhBMP-2 and C-rhBMP-2) and reference (BMPR1A, BMPR1B, and BMPR2) channel for 7 min at a flow rate of 10 µL/min.
The immobilization target level was set to 500 RU for each ligand (E-rhBMP-2 and C-rhBMP-2). E-rhBMP-2 stock samples and C-rhBMP-2 stock samples were diluted to 5.9 µg/mL and 0.5 µg/mL, respectively, in 10 mM sodium acetate (pH 6.0) buffer. The prepared ligand samples were only injected and immobilized on the sample channel. To deactivate the surface, 1 M ethanolamine (pH 8.5) was injected into both the sample and reference channels for 7 min at 10 µL/min.
All analyte samples (BMPR1A, BMPR1B, and BMPR2) were diluted in a concentration range of 10–2000 nM. Samples were injected into the sample and reference channels at a flow rate of 30 µL/min for an association phase of 120 s, followed by 120 s of dissociation to determine the association time, dissociation time, and concentration range of analysis. To test the regeneration conditions, NaOH in the concentration range of 10–50 mM was injected for 10 s at a flow rate of 30 µL/min. A 1:1 binding model describing one molecule of analyte binding to a single ligand molecule was used to calculate the KD.
C2C12 cell culture
Myoblastic C2C12 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). C2C12 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) at 37 °C and 5% CO2 humidified air using T75 flasks. When the cells reached confluence, they were used for other experiments. All reagents for cell culture were purchased from Gibco™ (Langley, VA, USA).
Western blot analysis
Cultured C2C12 cells were collected and seeded in 6-well plates (2 × 105 cells/well). The plates were then incubated at 37 °C and 5% CO2 for 24 h. E-rhBMP-2 and C-rhBMP-2 solutions were prepared at a concentration of 100 ng/mL by diluting with DMEM containing 2% bovine serum (BS). The prepared solutions were added to each well of a 6-well plate and incubated at 37 °C and 5% CO2. The cells were collected at 0, 3, and 6 h after rhBMP-2 treatment. The collected cells were lysed using a lysis buffer (50 mM Tris–HCl (pH 6.8), 2% SDS, 0.001% bromophenol blue, and 10% glycerol). The cell lysate was centrifuged (12,000×g) at 4 °C, after which the supernatant was recovered and protein concentration in the supernatant was measured using a BCA protein assay kit (Thermo Fisher Scientific Co) according to manufacturer instructions. Electrophoresis was carried out at 200 V for 30 min after loading 200 μg of protein into each well of 4–12% Bis–Tris Gels (Invitrogen, Carlsbad, CA, USA). The separated proteins from the gel were then transferred onto PVDF membranes (Thermo Fisher Scientific Co.). The membranes were blocked for 1 h with 5% skim milk, followed by reaction with anti-Phospho-Smad1/Smad5/Smad9 rabbit antibody (Danvers, MA, USA) diluted 1:1000 at 4 °C for 24 h. The membranes were then incubated with anti-rabbit HRP secondary antibody (Cambridge, MA, USA) diluted 1:2000 at 25 °C for 2 h. Peroxidase activity on the PVDF membrane was visualized by adding 3,3′,5,5′-Tetramethylbenzidine. Bands were detected after color development using a digital imaging system (Amersham Imager 600, GE Healthcare, Pittsburgh, PA, USA). The sampling process was repeated using primary antibodies against Smad1 and β-actin (Thermo Fisher Scientific Co.). Quantitation of the western blot was performed using GelAnalyzer 2010a software (http://www.gelanalyzer.com).
Cultured C2C12 cells were collected, and 2 mL of the cell solution (2 × 105 cells/mL) was seeded in 6-well plates. The cells were incubated at 37 °C and 5% CO2 for 24 h. E-rhBMP-2 and C-rhBMP-2 solutions were prepared at a concentration of 1000 ng/mL by diluting with DMEM containing 2% BS. After treating the cells with the prepared rhBMP-2 solutions, they were incubated at 37 °C and 5% CO2 and recovered after 18 and 24 h of incubation. After using the PureLink™ RNA mini kit (Invitrogen) to extract mRNA from the collected cells, a High-Capacity RNA-to-cDNA™ kit (Thermo Fisher Scientific Co.) was used to obtain cDNA.
The primers for osteogenic genes (Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN)) and β-actin gene (internal control) were purchased from Thermo Fisher Scientific Co. (Runx2: Mm00501580_m1, OCN: Mm03413826_mH, β-actin: Mm00607939_s1). PCR was performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), and the expression levels of Runx2 and OCN genes relative to β-actin were measured according to the manufacturer's instructions. For qRT-PCR analysis, experiments were performed in triplicate and values were expressed as the mean ± standard deviation (SD). Minitab 14 (Minitab Ltd, USA) was used to perform statistical analyses. Student's t-test was used to analyze differences in the expression level of the Runx2 and OCN genes between E-rhBMP-2 and C-rhBMP-2 at each time point. A value of P < 0.05 was considered significant.
Cultured C2C12 cells were collected, and the cell suspension was diluted with culture media to reach a concentration of 1 × 105 cells/mL. After adding 100 µL of the C2C12 cell solution (1 × 105 cells/mL) to each well of a 96-well plate, the cells were fixed by incubation at 37 °C and 5% CO2 for 24 h. E-rhBMP-2 and C-rhBMP-2 were prepared to a concentration of 5–1000 ng/mL (5.0, 23.3, 37.3, 59.6, 95.4, 152.6, 244.1, 390.6, 625.0, and 1000.0 ng/mL) by diluting with DMEM containing 2% BS. The prepared rhBMP-2 solutions at each concentration were used for treating the C2C12 cell solutions in triplicate in a 96-well plate. After 72 h of incubation, all solutions in the 96-well plate were removed and 100 µL of lysis buffer (100 mM glycine, 1 mM magnesium chloride, 1 mM zinc chloride, and 1% tergitol, pH 9.6) was added to each well to lyse the cells. Upon completion of lysis, 50 µL of p-nitrophenyl phosphate was added to each well followed by incubation at 25 °C for 30 min. Upon completion of the reaction, the absorbance at 405 nm was measured using a SpectraMax® M3 microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). Parallel-line assays were performed based on the measured dose–response curves using PLA 2.0 (Stegmann Systems GmbH, Germany). The experiments were performed on three separate plates with three replicates each.
Library construction and sequencing
Cultured C2C12 cells were collected and seeded in 6-well plates (2 × 105 cells/well). The plates were incubated at 37 °C and 5% CO2 for 24 h. E-rhBMP-2 and C-rhBMP-2 solutions were prepared at a concentration of 100 ng/mL by diluting with DMEM containing 2% BS. After treating each well of the 6-well plate with the prepared solutions, the plate was incubated at 37 °C and 5% CO2. The cells were collected after 3, 6, 12, and 24 h of incubation and mRNA was extracted as described in the qRT-PCR analysis section.
The acquired mRNA was purified using the Dynabeads® mRNA Purification Kit (Invitrogen) to deplete rRNA and enrich poly(A) + RNA using oligo d(T). Enriched mRNA was used for library construction using the MGIEasy RNA Directional Library Prep Kit (MGI, Shenzhen, China), according to the manufacturer’s instructions. The directional RNA libraries were then sequenced using the DNBSEQ-T7 sequencing instrument (BGI Genomics, China), according to the manufacturer’s instructions, yielding 150 bp paired-end reads. Sequencing results from this study have been deposited in the NCBI Short Read Archive under the accession number PRJNA812069.
Sequence alignment and gene expression analysis
Adapter sequence and low-quality bases were removed using the Cutadapt tool (version 2.9) (Martin 2011) and sequences under 36 bp were discarded by using the Trimmomatic tool (version 0.39) (Bolger et al. 2014). After trimming, reads were aligned to the mouse reference genome (mm10) and annotated by Ensembl v.102 using STAR (version 2.7.3a) with default parameters (Dobin et al. 2012). The read count and transcript per million values for individual transcripts were produced using the RSEM (version 1.3.1) software tool with default parameters (Li and Dewey 2011). The Bioconductor package edgeR was applied to identify differentially expressed genes (DEGs) with a P-value of 0.05 and fold change ≥ 2 criteria (Robinson et al. 2010).