Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

doi: 10.1074/jbc.m610929200

Figure Lengend Snippet: FIGURE 2. ProBMP1SSQQ binds BMP4 and BMP2 in a highly specific man- ner. A, an immunoblot is shown of samples containing () or lacking () 5 nM proBMP1SSQQ, BMP1, and/or BMP4; which were immunoprecipitated with anti-BMP1 antibodies (26). Blots were cut and the separate pieces were incu- bated with anti-BMP1 or anti-BMP4 antibodies. B, proBMP1SSQQ was incu- bated with a fixed amount of BMP4 (5 nM) alone, or in the presence of increas- ing amounts of BMP2 (molar ratios of BMP4:BMP2 of 1:1, 1:2, 1:5, and 1:10) followed by immunoprecipitation with anti-BMP1 antibody (26) and immu- noblotting with anti-BMP4 or anti-BMP2 monoclonal antibodies. As a control for pull-down efficiency, the anti-BMP4 blot was stripped and re-probed with anti-BMP1 antibodies to detect immunoprecipitated proBMP1SSQQ. C, puri- fied BMP1 prodomain, produced in a baculovirus system, was visualized by stainingwithCoomassieBrilliantBlueR-250(lane2).Numberstotheleftofgel correspond to the approximate sizes in kDa of molecular mass markers (lane 1). D, Western blot is used for characterization of anti-BMP prodomain anti- bodies, which at a 1:20,000 dilution detect 6 ng of BMP1-Fc fusion protein (lane 1), but do not detect 6 ng of Fc domain (lane 2).

Article Snippet: Recombinant BMP2, BMP4, BMP5, EGF, BMPR-1A/Fc, and TGF- 1 were all purchased from R&D systems.

Techniques: Western Blot, Immunoprecipitation, Bioprocessing, Control, Produced

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

doi: 10.1074/jbc.m610929200

Figure Lengend Snippet: FIGURE 3. The BMP1 prodomain directly binds BMP2 and BMP4 with high specificity and affinity. A, 5 nM BMP4 and His-tagged BMP1 prodomain were incubated separately or together, followed by precipitation with nickel- chargedaffinityresin(leftpanel)orwithanti-BMP1prodomainantibody(right panel) and immunoblot analysis with anti-prodomain or anti-BMP4 antibod- ies.B,5nMBMP2andHis-taggedBMP1prodomainwereincubatedseparately or together, followed by precipitation with anti-BMP1 prodomain antibody and protein A-Sepharose, and immunoblot analysis with anti-BMP1 prodo- main or with anti-BMP2 antibodies. C, 5 nM BMP4 and His-tagged BMP1 prodomain were coincubated, as in A, but in the presence of 10-fold excesses ofBMP2,TGF-1,EGF,orBMP5;followedbyprecipitationwithnickel-charged affinity resin and immunoblot analyses. D, 5 nM BMP4 and soluble BMP recep- tor IA (BMPR-1A, ALK-3), fused to an Fc domain, were coincubated in the presence of 1:1, 2:1, or 5:1 molar ratios of either BMP1 prodomain or Chordin, and the samples then precipitated with protein A-Sepharose and analyzed by immunoblots employing anti-BMP4 or anti-BMPR-1A antibodies.

Article Snippet: Recombinant BMP2, BMP4, BMP5, EGF, BMPR-1A/Fc, and TGF- 1 were all purchased from R&D systems.

Techniques: Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

doi: 10.1074/jbc.m610929200

Figure Lengend Snippet: FIGURE 4. The BMP1 prodomain binds BMP2 with a KD of 10.9 nM. Coomassie Brilliant Blue R-250-stained SDS-PAGE gels are shown for molecular mass standards (lane 1) and purified BMP1 prodomain-Fc fusion protein (lane 2) (A) or molecular mass standards (lane 1) and purified BMP1 prodomain minus the Fc domain (lane 2) (B). Numbers to the left of gels correspond to the approximate sizes in kDa of molecular mass markers. BMP1 prodomain (C) and murine Chordin (D) bind to BMP2 with KDs of 10.9 4.7 nM and 6.7 1.0 nM, respectively.

Article Snippet: Recombinant BMP2, BMP4, BMP5, EGF, BMPR-1A/Fc, and TGF- 1 were all purchased from R&D systems.

Techniques: Staining, SDS Page, Purification

Journal: Clinical and Experimental Dental Research

Article Title: Absorbable collagen sponges loaded with recombinant bone morphogenetic protein 9 induces greater osteoblast differentiation when compared to bone morphogenetic protein 2

doi: 10.1002/cre2.55

Figure Lengend Snippet: Growth factor adsorption of recombinant human bone morphogenetic protein 2 and recombinant human bone morphogenetic protein 9to absorbable collagen sponges at 0 min, 15 min, 60 min, 8 hrs, 24 hrs, 3 days, and 10 days as quantified by enzyme‐linked immunosorbent assay. absorbable collagen sponges were able to efficiently adsorb recombinant bone morphogenetic protein 2 and bone morphogenetic protein 9 and have comparable release kinetics over time

Article Snippet: Recombinant human BMP2 and rhBMP9 were purchased from R&D systems Inc (Minneapolis, MM, USA).

Techniques: Adsorption, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Dental Research

Article Title: Absorbable collagen sponges loaded with recombinant bone morphogenetic protein 9 induces greater osteoblast differentiation when compared to bone morphogenetic protein 2

doi: 10.1002/cre2.55

Figure Lengend Snippet: (a) Attachment (8 hrs) and (b) proliferation (1, 3, and 5 days) assays of ST2 cells seeded on control tissue culture plastic, control absorbable collagen sponges (ACS), ACS loaded with bone morphogenetic protein 2 low (10 ng/ml), ACS loaded with bone morphogenetic protein 2 high (100 ng/ml), ACS loaded with bone morphogenetic protein 9 low (10 ng/ml), and ACS loaded with bone morphogenetic protein 9 high (100 ng/ml). No significant difference was observed between six groups at all time points

Article Snippet: Recombinant human BMP2 and rhBMP9 were purchased from R&D systems Inc (Minneapolis, MM, USA).

Techniques: Control

Journal: Clinical and Experimental Dental Research

Article Title: Absorbable collagen sponges loaded with recombinant bone morphogenetic protein 9 induces greater osteoblast differentiation when compared to bone morphogenetic protein 2

doi: 10.1002/cre2.55

Figure Lengend Snippet: (a) Alkaline phosphatase staining of absorbable collagen sponges (ACS) at 7 days of ST2 cells seeded on control tissue culture plastic, control ACS, ACS loaded with BMP2 low (10 ng/ml), ACS loaded with BMP2 high (100 ng/ml), ACS loaded with BMP9 low (10 ng/ml), and ACS loaded with BMP9 high (100 ng/ml). (b) bone morphogenetic protein 9 low and high significantly increased alkaline phosphatase staining when compared to control and bone morphogenetic protein 2samples (* denotes significant difference, p < .05; ** denotes significantly higher than all other treatment modalities, p < .05)

Article Snippet: Recombinant human BMP2 and rhBMP9 were purchased from R&D systems Inc (Minneapolis, MM, USA).

Techniques: Staining, Control

Journal: Clinical and Experimental Dental Research

Article Title: Absorbable collagen sponges loaded with recombinant bone morphogenetic protein 9 induces greater osteoblast differentiation when compared to bone morphogenetic protein 2

doi: 10.1002/cre2.55

Figure Lengend Snippet: Real‐time PCR of ST2 cells seeded on control tissue culture plastic, control absorbable collagen sponges (ACS), ACS loaded with BMP2 low (10 ng/ml), ACS loaded with BMP2 high (100 ng/ml), ACS loaded with BMP9 low (10 ng/ml), and ACS loaded with BMP9 high (100 ng/ml) for genes encoding (a) runt‐related transcription factor 2, (b) collagen 1 alpha 2 (COL1a2), (c) alkaline phosphatase (ALP), (d) bone sialoprotein (BSP), and (e) osteocalcin (OCN) at 3 and 14 days postseeding (* denotes significant difference, p < .05; ** denotes significantly higher than all other treatment modalities, p < .05)

Article Snippet: Recombinant human BMP2 and rhBMP9 were purchased from R&D systems Inc (Minneapolis, MM, USA).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Clinical and Experimental Dental Research

Article Title: Absorbable collagen sponges loaded with recombinant bone morphogenetic protein 9 induces greater osteoblast differentiation when compared to bone morphogenetic protein 2

doi: 10.1002/cre2.55

Figure Lengend Snippet: (a) Visual representation of alizarin red‐stained of negative control absorbable collagen sponges (ACS) without cells, control tissue culture plastic, control ACS, ACS loaded with BMP2 low (10 ng/ml), ACS loaded with BMP2 high (100 ng/ml), ACS loaded with BMP9 low (10 ng/ml), and ACS loaded with BMP9 high (100 ng/ml) at 14 days postseeding. Note the intensity of red staining of ACS coated with BMP9 in comparison to control and BMP2 samples. (b) Quantified data of alizarin red staining from colour thresholding software (* denotes significant difference, p < .05; ** denotes significantly higher than all other treatment modalities, p < .05)

Article Snippet: Recombinant human BMP2 and rhBMP9 were purchased from R&D systems Inc (Minneapolis, MM, USA).

Techniques: Staining, Negative Control, Control, Comparison, Software

Journal: Communications Biology

Article Title: PiR48444 inhibits MSC osteogenic differentiation and bone regeneration via targeting METTL7A/eIF4E-mediated BMP2 m6A methylation

doi: 10.1038/s42003-026-09583-1

Figure Lengend Snippet: A Alizarin red staining at day 14. B Expression pattern of osteogenic differentiation markers at day 7. C The correlation analysis heatmap of differentially expressed piRNAs and mRNAs in SHED at day 7 of osteogenic differentiation. D Differentially expressed piRNAs targeting BMP2 in SHED. E The bioinformatic expression pattern of piR48444. F The RT-qPCR analysis of piR48444 in different MSCs ( G ), LPS-treated BMSCs ( H ), and aged BMSCs ( I ). The bioinformatics analysis of piR48444 expression in mononuclear cells from peripheral blood ( n = 3) ( GSE159121 ). J RT-qPCR analysis of piR48444 expression in plasma ( n = 15) of osteoporosis patients. K The cytoplasm and nucleus separation analysis of piR48444 in the nucleus and cytoplasm. L The piR48444 probe staining. M The binding of piR48444 with PIWIL1 protein. N The expression level of piR48444 in PIWIL1 -KD SHED. Data in ( B , E – J , L , N ) presented as mean ± SD; statistical analysis via Student’s t test for two-group comparisons; significance levels: * p < 0.05, ** p < 0.01 and *** p < 0.001. PM proliferation medium; OM osteogenic medium.

Article Snippet: Then PVDF membranes were incubated with the primary antibodies against METTL7A (Proteintech, cat#17092-1-AP, 1:5,000, China), BMP2 (Proteintech, cat#6383-1-Ig, 1:5000, China), PIWIL1 (Zenbio, cat#R382334, 1:5000, China), PIWIL2 (SAB, cat#25385, 1:5000, USA), PIWIL4 (SAB, cat#31113, 1:5000, USA) and GAPDH (Proteintech, cat#10494-1-AP, 1:10,000, China) overnight at 4 °C.

Techniques: Staining, Expressing, Quantitative RT-PCR, Clinical Proteomics, Binding Assay

Journal: Communications Biology

Article Title: PiR48444 inhibits MSC osteogenic differentiation and bone regeneration via targeting METTL7A/eIF4E-mediated BMP2 m6A methylation

doi: 10.1038/s42003-026-09583-1

Figure Lengend Snippet: A Predicated target mRNAs of piR48444 in SHED. B PiR48444 expression negatively correlated with METTL7A , BMP2 expression ( n = 3). C , D The expression pattern and RT-qPCR analysis of METTLA , BMP2 . E , F METTL7A and BMP2protein expression patterns in piR48444-OE and piR48444-KD SHED ( n = 3). G IHC for METTL7A and BMP2 in piR48444 knockdown cell sheets for calvarial defect repair. H Luciferase reporter assay validation of piR48444 targets METTL7A and BMP2 ( n = 4). Data are presented as mean ± SD. Statistical significance was determined by Student’s t test ( C , D , F ) and one-way ANOVA ( H ). Significance levels: * p < 0.05 and ** p < 0.01, and *** p < 0.001. OE overex p ression, KD knockdown, Wt wildtype, Mu mutant.

Article Snippet: Then PVDF membranes were incubated with the primary antibodies against METTL7A (Proteintech, cat#17092-1-AP, 1:5,000, China), BMP2 (Proteintech, cat#6383-1-Ig, 1:5000, China), PIWIL1 (Zenbio, cat#R382334, 1:5000, China), PIWIL2 (SAB, cat#25385, 1:5000, USA), PIWIL4 (SAB, cat#31113, 1:5000, USA) and GAPDH (Proteintech, cat#10494-1-AP, 1:10,000, China) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Luciferase, Reporter Assay, Biomarker Discovery, Mutagenesis

Journal: Communications Biology

Article Title: PiR48444 inhibits MSC osteogenic differentiation and bone regeneration via targeting METTL7A/eIF4E-mediated BMP2 m6A methylation

doi: 10.1038/s42003-026-09583-1

Figure Lengend Snippet: A , B METTL7A and BMP2 protein expression in METTL7A -OE and METTL7A -KD SHED. C BMP2 mRNA expression in METTL7A -OE and METTL7A -KD SHED. D Immunostaining and quantification for METTL7A in SHED. E METTL7A expression in the cytoplasm and nucleus. F The direct binding of METTL7A with BMP2 mRNA by RIP assay. G BMP2 m 6 A modification level in METTL7A -OE and METTL7A -KD SHED. H BMP2 mRNA stability in METTL7A -OE and METTL7A -KD SHED. BMP2 protein synthesis in METTL7A -OE ( I, J) and METTL7A -KD ( K, L ) SHED. BMP2 protein degradation in METTL7A -OE ( M , N ) and METTL7A -KD ( O , P ) SHED. Data are presented as mean ± SD. Data are presented as mean ± SD. Statistical significance was determined by Student’s t test ( B , C , D , F , G ), two-way ANOVA ( H ), and one-way ANOVA ( J , L , N , P ). Significance levels: * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 3. OE overexpression, KD knockdown.

Article Snippet: Then PVDF membranes were incubated with the primary antibodies against METTL7A (Proteintech, cat#17092-1-AP, 1:5,000, China), BMP2 (Proteintech, cat#6383-1-Ig, 1:5000, China), PIWIL1 (Zenbio, cat#R382334, 1:5000, China), PIWIL2 (SAB, cat#25385, 1:5000, USA), PIWIL4 (SAB, cat#31113, 1:5000, USA) and GAPDH (Proteintech, cat#10494-1-AP, 1:10,000, China) overnight at 4 °C.

Techniques: Expressing, Immunostaining, Binding Assay, Modification, Over Expression, Knockdown

Journal: Communications Biology

Article Title: PiR48444 inhibits MSC osteogenic differentiation and bone regeneration via targeting METTL7A/eIF4E-mediated BMP2 m6A methylation

doi: 10.1038/s42003-026-09583-1

Figure Lengend Snippet: A , B Bioinformatic and RT-qPCR analysis of eIF4EBP1 and eIF4E in SHED during osteogenic differentiation. C The expression of eIF4EBP1 and eIF4E in piR48444-KD SHED. D The binding prediction analysis of METTL7A and eIF4E by ClusPro and PyMOL. E Co-IP analysis of the binding between METTL7A and eIF4E. F Expression pattern of eIF4E in eIF4E -OE SHED. G The expression levels of osteogenic genes, including ALP , RUNX2 , and OCN , at day 7. H The staining of ALP and quantification at day 7. I Expression pattern of eIF4E in eIF4E -KD SHED at day 7. J The expression levels of osteogenic genes, including ALP , RUNX2 , and OCN , at day 7. K The staining of ALP and quantification at day 7. L, M BMP2 expression and quantification in eIF4E -OE and eIF4E -KD SHED. N The expression levels of osteogenic genes, including ALP , RUNX2 , and OCN , at day 7. O The staining of ALP and quantification at day 7. Data are presented as mean ± SD ( n = 3). Statistical significance was determined by Student’s t test ( A – C , F , I , M ), and one-way ANOVA ( G , H , J , K , N , O ). Significance levels: * p < 0.05, ** p < 0.01, and *** p < 0.001. OE overex p ression, KD knockdown.

Article Snippet: Then PVDF membranes were incubated with the primary antibodies against METTL7A (Proteintech, cat#17092-1-AP, 1:5,000, China), BMP2 (Proteintech, cat#6383-1-Ig, 1:5000, China), PIWIL1 (Zenbio, cat#R382334, 1:5000, China), PIWIL2 (SAB, cat#25385, 1:5000, USA), PIWIL4 (SAB, cat#31113, 1:5000, USA) and GAPDH (Proteintech, cat#10494-1-AP, 1:10,000, China) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Co-Immunoprecipitation Assay, Staining, Knockdown