Journal: JBMR Plus

Article Title: Establishment and characterization of a human pre-osteocyte cell line: hOsteo4-E9

doi: 10.1093/jbmrpl/ziaf163

Figure Lengend Snippet: Characterization of human-derived hOsteo4 subclones. (A) Phase contrast images of 4 hOsteo4 subclones, that is, C7, D8, E9, and C10; (B) cell viability test by CCK8 assay for 4 hOsteo4 subclones and MLO-Y4 cells; N = 3 for each group; (C) immunofluorescence staining of DMP1, FGF23, and sclerostin of 4 hOsteo4 subclones, MLO-Y4 and MC3T3 cells; IgG group were MC3T3 cells and served as a negative background control; (D) western-blot results for Dmp1, Sclerostin, E11, integrin β1, integrin β3, ALP, and Col-I protein expressions from hOsteo4 subclones, MLO-Y4 cells and MC-3 T3 cells; tubulin served as a loading control.

Article Snippet: Integrin β3 , CST , 13166S , 1:1000 , .

Techniques: Derivative Assay, CCK-8 Assay, Immunofluorescence, Staining, Control, Western Blot

Journal: JBMR Plus

Article Title: Establishment and characterization of a human pre-osteocyte cell line: hOsteo4-E9

doi: 10.1093/jbmrpl/ziaf163

Figure Lengend Snippet: Subclone hOsteo4-E9 cells underwent dramatic morphological changes upon FSS treatment. (A) Phase contrast images showed the morphological changes of hOsteo4 subclones in static and FSS conditions; (B) quantitative analysis of percentages of dendritic cells of 4 hOsteo4 subclones with and without FSS; N = 3 for each group; (C) WB results of p-FAK, integrin β3, and kindlin-2 protein changes of hOsteo4-E9 subclone under different FSS stimuli; (D) phase contrast images of cell shape changes of hOsteo4-E9 subclone under different FSS stimuli; (E-G) statistical analysis of percentages of dendritic cells, number of dendrites per cell and the longest dendritic length per cell of hOsteo4-E9 subclone under FSS stimuli; quantitative results were analyzed from 3 independent experiments; (H) IF staining of F-actin cytoskeleton and DAPI nuclei in hOsteo4-E9 cells with and without (static) FSS stimuli; (I) IF images stained with F-actin, p-FAK, tubulin, and Cx43 in hOsteo4-E9 cells with and without (static) FSS stimuli. Arrowheads in figures indicated the direction of FSS forces. Results are expressed as mean ± SD. n. s. p > .05; * p < .05; ** p < .01; *** p < .001.

Article Snippet: Integrin β3 , CST , 13166S , 1:1000 , .

Techniques: Staining

Journal: JBMR Plus

Article Title: Establishment and characterization of a human pre-osteocyte cell line: hOsteo4-E9

doi: 10.1093/jbmrpl/ziaf163

Figure Lengend Snippet: hOsteo4-E9 cells showed cytoskeleton remodeling with enhanced proteins expressions of mechanosensitive genes similar to MLO-Y4 cells in response to FSS treatment. (A) Phase contrast images of MLO-Y4 and hOsteo4-E9 cells under static/0, 1.5, and 3.5 dynes/cm 2 FSS; (B-E) quantitative analysis of dendritic percentages, number of dendrites per cell, the longest dendritic length in single cell and spreading area changes in MLO-Y4 and hOsteo4-E9 cells under different FSS stimuli; (F) SEM images of MLO-Y4 and hOsteo4-E9 cells with or without FSS; (G) F-actin staining of MLO-Y4 and hOsteo4-E9 cells with or without FSS; (H, I) quantitative analysis of the length of secondary dendrites and the number of secondary dendrites per cell in MLO-Y4 and hOsteo4-E9 cells with or without FSS; (J) western-blot results for p-FAK, kindlin-2, integrin β3, Cx43, Dmp1, and Sclerostin in MLO-Y4, and hOsteo4-E9 cells under different FSS stimuli; tubulin served as a loading control; (K-P) statistical analysis of protein expressions of p-FAK, kindlin-2, integrin β3, Cx43, Dmp1, and Sclerostin in MLO-Y4 and hOsteo4-E9 cells under different FSS stimuli. N = 3 for each group. Arrowheads in figures indicated the direction of FSS forces. Results are expressed as mean ± SD. n.s. p > .05; * p < .05; ** p < .01; *** p < .001.

Article Snippet: Integrin β3 , CST , 13166S , 1:1000 , .

Techniques: Single Cell, Staining, Western Blot, Control

Journal: JBMR Plus

Article Title: Establishment and characterization of a human pre-osteocyte cell line: hOsteo4-E9

doi: 10.1093/jbmrpl/ziaf163

Figure Lengend Snippet: RNA-sequencing data revealed that hOsteo4-E9 cells not only share some similarity with MLO-Y4 cells, but also have distinct transcription profiles in response to FSS. (A) PCA plot of gene expression variability of human hOsteo4-E9, murine MLO-Y4, and murine long bone-derived osteocytes; (B) Venn diagram of detected genes in hOsteo4-E9 and MLO-Y4 cells; (C) Venn diagram of detected genes in 4 experimental groups, that is, E9_F, E9_S, Y4_F, and Y4_S; (D-F) top 15 of KEGG enriched pathways in all detected genes, hOsteo4-E9 specific and MLO-Y4 specific genes; (G) clustering heat map highlighted the variations of FA-associated gene expression patterns between 4 experimental groups; (H-M) qPCR validation of gene expression of integrin αν, integrin α5, integrin α3, integrin β5, integrin β7, and EMP1 in 4 experimental groups. N = 3 for each group. Results are expressed as mean ± SD. n.s. p > .05; * p < .05; ** p < .01; *** p < .001.

Article Snippet: Integrin β3 , CST , 13166S , 1:1000 , .

Techniques: RNA Sequencing, Gene Expression, Derivative Assay, Biomarker Discovery

Journal: Bioactive Materials

Article Title: Designed bone-targeting ROS-responsive nanoplatform for precision glycolysis inhibition in postmenopausal osteoporosis

doi: 10.1016/j.bioactmat.2025.11.032

Figure Lengend Snippet: AZD3965 inhibits key osteoclast marker protein expression in vitro. (A) Protein expression of integrin-β3, NFATc1, MMP9, CTSK and c-Fos detected via Western blotting after 5-day treatment with RANKL and AZD3965 (0, 5, 10 μM). (B–F) Quantification of osteoclast marker protein expression with specified intervention (all normalized to β-actin). (G) NFATc1 expression and nuclear translocation detected via immunofluorescence staining (scale bar = 50 μm). (H) Quantification of NFATc1 mean fluorescence intensity in the nucleus. (I) Protein expression of integrin-β3, NFATc1, MMP9, CTSK and c-Fos detected via Western blotting after treatment with RANKL and AZD3965 at different time points. (J–N) Quantification of osteoclast marker protein expression on the specified day (all normalized to β-actin). N = 3. ##P < 0.01, ###P < 0.001, ####P < 0.0001, compared to control group. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001, compared to the RANKL alone treatment group.

Article Snippet: Antibodies against osteoclast marker proteins, such as c-Fos, CTSK, integrin β3, and NFATc1, were procured from Santa Cruz (San Jose, United States).

Techniques: Marker, Expressing, In Vitro, Western Blot, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Control

Journal: Bioactive Materials

Article Title: Designed bone-targeting ROS-responsive nanoplatform for precision glycolysis inhibition in postmenopausal osteoporosis

doi: 10.1016/j.bioactmat.2025.11.032

Figure Lengend Snippet: PH/DPA@A inhibits RANKL-induced osteoclast formation and bone resorption function in vitro. (A) Protein expression of integrin-β3, NFATc1, MMP9, CTSK and c-Fos detected via Western blotting after 5-day treatment with RANKL and PH/DPA@A in five groups. (B–F) Quantification of osteoclast marker protein expression with indicated intervention (all normalized to β-actin). (G) TRAcP staining to assess osteoclast differentiation of BMMs in five groups. (H) Quantification of TRAcP staining in five groups. (I, J) F-actin staining and quantification of F-actin ring area in five groups. (K–L) Bone slice assay using mature osteoclasts and quantification of resorption area in five groups. Scale bar = 200 μm. N = 3. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

Article Snippet: Antibodies against osteoclast marker proteins, such as c-Fos, CTSK, integrin β3, and NFATc1, were procured from Santa Cruz (San Jose, United States).

Techniques: In Vitro, Expressing, Western Blot, Marker, Staining

Journal: Journal of Functional Biomaterials

Article Title: LXW7 Peptide Modification of Acellular Liver Scaffolds Improves Endothelialization and Hemocompatibility in Bioengineered Liver

doi: 10.3390/jfb17030122

Figure Lengend Snippet: Thrombogenicity evaluation of endothelialized scaffolds ( A ) schematic of the experimental design of ex vivo blood perfusion. ( B ) Gross appearance of DLS, endothelialized, and LXW7 endothelialized scaffolds after blood perfusion, showing markedly reduced clot deposition in the LXW7 group (yellow circles highlight thrombi). Scale bar = 2 cm. ( C ) IF staining of platelet marker integrin αIIb (red) with DAPI (blue) showing reduced platelet adhesion in endothelialized and LXW7endothelialized scaffolds compared to DLS. Scale bar = 100 µm. ( D ) Quantification of fluorescence intensity confirming significantly decreased platelet adhesion in LXW7 endothelialized scaffolds ( n = 4 fields, * p < 0.05 vs. DLS). ( E ) Time-dependent changes in platelet count (%) in the blood perfusate during ex vivo blood perfusion ( n = 3 samples each time point, * p < 0.05 vs. DLS).

Article Snippet: For IF staining, sections were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 2% bovine serum albumin (Sigma-Aldrich) for 45 min. Tissue sections were incubated overnight with primary antibodies including anti-human CD31 (1:100, MA5-13188, Invitrogen), Albumin (1:200, PA5-89332, Invitrogen), anti-Integrin αIIb (1:100, sc-21783, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-TGF-β1 (1:200, ab170874, Abcam).

Techniques: Ex Vivo, Staining, Marker, Fluorescence