Journal: Clinical Implant Dentistry and Related Research

Article Title: Bone Morphogenetic Protein ( BMP ) 9 Outperforms BMP2 in Osteogenesis and Osseointegration: In Vitro and In Vivo

doi: 10.1111/cid.70135

Figure Lengend Snippet: Comparative effects of BMP9 and BMP2 on osteogenic differentiation and osteoclastogenesis in vitro. (A) Real‐time PCR analysis of key osteogenic genes (Col1, Runx2, ALP, and OCN) in MC3T3‐E1 cells treated with 8 nM of BMP2 or BMP9 for 3, 5, and 7 days. All gene‐expression levels were normalized to GAPDH. (B) Western blot analysis of osteogenic marker proteins in cell lysates harvested after 7 days of treatment with BMP2 or BMP9. GAPDH was used as the loading control. Densitometric quantification of band intensities (integrated density) normalized to GAPDH is shown below the blots and presented as relative protein expression. (C) Western blot showing dose‐dependent p‐Smad1/5/9 in MC3T3‐E1 cells exposed to varying concentrations of BMP2 or BMP9. Phosphorylation was quantified by densitometry and expressed as fold change vs. control after normalization using [(p‐Smad1/5/9)/(total Smad1/5/9)] and further normalized to GAPDH, as shown in the graph below the blots. Asterisks indicate statistical significance for pairwise comparisons between BMP2 and BMP9 at the same concentration (****, p < 0.0001), unless otherwise indicated. (D) ALP activity and representative images of ALP staining in MC3T3‐E1 cultures after 7 days of induction with BMP2 or BMP9. (E) Alizarin Red S staining illustrating mineralized nodule formation after extended culture with BMP2 or BMP9. (F) Representative TRAP‐stained images of RAW 264.7‐derived osteoclasts following treatment with RANKL (3 nM), BMP2 (8 nM), or BMP9 (8 nM) for 5 days. TRAP‐positive multinucleated osteoclasts are indicated by arrows. Scale bar, 20 μm. (G) Quantification of TRAP‐positive multinucleated cells per well. Data are presented as the mean ± SD ( n = 3 independent experiments), and p ‐values were calculated using one‐way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). BMP, bone morphogenetic protein; PCR, polymerase chain reaction; ALP, alkaline phosphatase; Col1, collagen type I; Runx2, runt‐related transcription factor 2; OCN, osteocalcin; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Recombinant human RANKL (11682‐HNCH; Sino Biological, Beijing, China) and a tartrate‐resistant acid phosphatase (TRAP) staining kit (MK300; Takara Bio, Shiga, Japan) were used for the osteoclast differentiation assay.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Marker, Control, Expressing, Phospho-proteomics, Concentration Assay, Activity Assay, Staining, Derivative Assay, Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Pla2g7 regulates bone homeostasis via Alox12/12-HETE/Gpr31 signaling axis

doi: 10.1038/s41467-025-66285-8

Figure Lengend Snippet: A TRAP and F-actin staining in BMMs derived from Pla2g7 f/f and Pla2g7 LysM mice following osteoclast induction (scale bar, normal, 500 μm, enlarged, 200 μm). B Quantification of TRAP positive MNCs and average osteoclast size. n = 6 biologically independent wells per group. C , D Relative gene and protein expression of osteoclast markers, and the densitometric analysis of Western blot bands. RNA, n = 3 biologically independent wells per group. WB, n = 3 biologically independent cell samples per group. E The molecular structure diagram of Darapladib. F , G TRAP and F-actin staining and quantification of osteoclasts stimulated with RANKL in the absence or presence of Darapladib in a concentration-dependent manner (0, 100, 200, and 400 nM), with Darapladib administered throughout the entire induction period. n = 6 biologically independent wells per group. (scale bar, normal, 500 μm, enlarged, 200 μm). H , I The gene and protein expression of osteoclast markers. n = 3 biologically independent wells per group. J The immunoblotting analysis in osteoclasts induced by RANKL with or without Darapladib at different time points. K , L Hydroxyapatite resorption assay and statistical analysis of BMMs incubated with RANKL with or without Darapaldib (400 nmol/L). n = 3 biologically independent cell samples per group. (scale bar, normal, 200 μm). M TRAP and F-actin staining images of osteoclasts stimulated by RANKL in the absence or presence of recombinant Pla2g7 protein (50, 100, or 200 ng/mL) for 5 days, with Pla2g7 administered throughout the entire induction period (scale bar, normal, 500 μm, enlarged, 200 μm). N , O Relative gene and protein expression of osteoclast-specific markers. n = 3 biologically independent wells per group. P Western blot analysis of BMMs incubated with RANKL in the absence or presence of Pla2g7 at various time points. Data were showed as mean ± SD. Two-sided Student’s t test ( B – D , L ) was employed to assess the difference between the two groups. One-way ANOVA ( G ) and two-way ANOVA ( H , N ) were utilized for multiple comparisons test. Significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns non-significance.

Article Snippet: Recombinant mouse M-CSF (Cat#CB34, Novoprotein; Cat#AM10003, Amizona Scientific) and RANKL (Cat#462-TEC, R&D System), as well as recombinant human M-CSF (Cat#11682-HNCH, SinoBiological) and RANKL (Cat#11792-HNAH, SinoBiological).

Techniques: Staining, Derivative Assay, Expressing, Western Blot, Concentration Assay, Incubation, Recombinant

Journal: Nature Communications

Article Title: Pla2g7 regulates bone homeostasis via Alox12/12-HETE/Gpr31 signaling axis

doi: 10.1038/s41467-025-66285-8

Figure Lengend Snippet: A The design of lipid metabolomics assay, in which Darapladib treatment was conducted concurrently with RANKL induction for 3 days until osteoclasts emerged. n = 5 biologically independent cell samples per group. B Volcano plot indicating differentially expressed genes in BMMs incubated with RANKL in the absence or presence of Darapladib (400 nmol/L). C Network analysis of the top 5 most significantly upregulated and downregulated secondary metabolites according to the integrated analysis of Fold change and P value. D Heatmap of differentially expressed secondary metabolites. FFA free fatty acid, SM sphingomyelin, LPC lysophosphatidylcholine, LPI lysophosphatidylinositol, LPE lysophosphatidylethanolamine, PC phosphatidylcholine, PS phosphatidylserine, PI phosphatidylinositol, PG phosphatidylglycerol, PE phosphatidylethanolamine. n = 5 biologically independent cell samples per group. E A schematic diagram of the RNA-sequencing of BMMs from WT mice with or without Darapladib, following 3 days of RANKL induction until osteoclasts emerged. n = 3 biologically independent cell samples per group. F Volcano plot showing 1036 upregulated and 1979 downregulated genes for RNA-seq data. G A heatmap of osteoclast-specific genes between RANKL and RANKL treated by Darapladib groups. n = 3 biologically independent cell samples per group. H The metabolism class in KEGG analysis of RNA-seq data. I , J The concentration of arachidonic acid in Darapladib, recombinant Pla2g7 and Pla2g7 cKO groups. n = 4 biologically independent mice per group. K – M The expression of ferroptosis-related proteins in Darapladib, recombinant Pla2g7 and Pla2g7 cKO groups, and these densitometric analysis of Western blot bands. n = 3 biologically independent cell samples per group. Data were showed as mean ± SD. Two-sided Student’s t test ( I , J and K – M ) was employed to assess the difference between the two groups. Significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns non-significance.

Article Snippet: Recombinant mouse M-CSF (Cat#CB34, Novoprotein; Cat#AM10003, Amizona Scientific) and RANKL (Cat#462-TEC, R&D System), as well as recombinant human M-CSF (Cat#11682-HNCH, SinoBiological) and RANKL (Cat#11792-HNAH, SinoBiological).

Techniques: Incubation, RNA Sequencing, Concentration Assay, Recombinant, Expressing, Western Blot