Journal: Frontiers in Cell and Developmental Biology

Article Title: Localized sclerostin accumulation in osteocyte lacunar-canalicular system is associated with cortical bone microstructural alterations and bone fragility in db/db male mice

doi: 10.3389/fcell.2025.1562764

Figure Lengend Snippet: Reduced osteoblast and osteoclast activity in 20-week-old male db/db diabetic mice. H&E (A) and TRAP (B) staining show reduced activity of both osteoblasts and osteoclasts in db/db mice (n = 5 per group). (C) Serum analysis shows lower osteocalcin, TRACP 5b, and CTX-1 levels, and higher sclerostin levels in db/db mice (n = 4-5 per group). Data are presented as mean ± SD, analyzed by unpaired Student’s t-test. *p < 0.05, **p < 0.01.

Article Snippet: For recombinant sclerostin (SCL) treatment, IDG-SW3 cells were treated with 100 ng/mL recombinant mouse sclerostin (HY- P70717 , MCE, China) for 48 h following 28 days of osteogenic induction.

Techniques: Activity Assay, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Localized sclerostin accumulation in osteocyte lacunar-canalicular system is associated with cortical bone microstructural alterations and bone fragility in db/db male mice

doi: 10.3389/fcell.2025.1562764

Figure Lengend Snippet: Localized sclerostin accumulation and increased expression of PLR-related proteins in 20-week-old male db/db diabetic mice cortical bone. Immunohistochemical staining shows increased expression of CTSK (A) , MMP-13 (B) , and sclerostin (C) within the osteocyte LCS of db/db mice compared to WT mice. Linear regression analysis (D) reveals a significant positive correlation between sclerostin expression and CTSK and MMP-13 using combined data from both groups. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired Student’s t-test for (A–C) (n = 5 mice per group) and Pearson correlation for (D) . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For recombinant sclerostin (SCL) treatment, IDG-SW3 cells were treated with 100 ng/mL recombinant mouse sclerostin (HY- P70717 , MCE, China) for 48 h following 28 days of osteogenic induction.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Localized sclerostin accumulation in osteocyte lacunar-canalicular system is associated with cortical bone microstructural alterations and bone fragility in db/db male mice

doi: 10.3389/fcell.2025.1562764

Figure Lengend Snippet: Effects of high glucose and recombinant Sclerostin (SCL) on osteocyte remodeling markers in IDG-SW3 cells. (A) Representative images showing osteocytic differentiation over 28 days, including DMP1-GFP fluorescence, ALP staining, and ARS staining. (B) Time-course expression of late osteocytic markers (FGF23, MEPE, Pdpn, Phex, and Sost) over 28 days, presented as relative fold induction. (C) Elevated mRNA expression of Sost, Ctsk, Mmp-13, and Atp6v0d2 in IDG-SW3 cells treated with 20 mM glucose compared to control cells. (D) Upregulation of Ctsk and Mmp-13 mRNA levels in IDG-SW3 cells treated with recombinant SCL compared to vehicle control. Data represent mean ± SD from three independent experiments (n = 3). Statistical significance is indicated (*p < 0.05, **p < 0.01, ****p < 0.0001).

Article Snippet: For recombinant sclerostin (SCL) treatment, IDG-SW3 cells were treated with 100 ng/mL recombinant mouse sclerostin (HY- P70717 , MCE, China) for 48 h following 28 days of osteogenic induction.

Techniques: Recombinant, Fluorescence, Staining, Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Localized sclerostin accumulation in osteocyte lacunar-canalicular system is associated with cortical bone microstructural alterations and bone fragility in db/db male mice

doi: 10.3389/fcell.2025.1562764

Figure Lengend Snippet: Reduced osteoblast and osteoclast activity in 20-week-old male db/db diabetic mice. H&E (A) and TRAP (B) staining show reduced activity of both osteoblasts and osteoclasts in db/db mice (n = 5 per group). (C) Serum analysis shows lower osteocalcin, TRACP 5b, and CTX-1 levels, and higher sclerostin levels in db/db mice (n = 4-5 per group). Data are presented as mean ± SD, analyzed by unpaired Student’s t-test. *p < 0.05, **p < 0.01.

Article Snippet: For recombinant sclerostin (SCL) treatment, IDG-SW3 cells were treated with 100 ng/mL recombinant mouse sclerostin (HY- P70717 , MCE, China) for 48 h following 28 days of osteogenic induction.

Techniques: Activity Assay, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Localized sclerostin accumulation in osteocyte lacunar-canalicular system is associated with cortical bone microstructural alterations and bone fragility in db/db male mice

doi: 10.3389/fcell.2025.1562764

Figure Lengend Snippet: Localized sclerostin accumulation and increased expression of PLR-related proteins in 20-week-old male db/db diabetic mice cortical bone. Immunohistochemical staining shows increased expression of CTSK (A) , MMP-13 (B) , and sclerostin (C) within the osteocyte LCS of db/db mice compared to WT mice. Linear regression analysis (D) reveals a significant positive correlation between sclerostin expression and CTSK and MMP-13 using combined data from both groups. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired Student’s t-test for (A–C) (n = 5 mice per group) and Pearson correlation for (D) . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For recombinant sclerostin (SCL) treatment, IDG-SW3 cells were treated with 100 ng/mL recombinant mouse sclerostin (HY- P70717 , MCE, China) for 48 h following 28 days of osteogenic induction.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Localized sclerostin accumulation in osteocyte lacunar-canalicular system is associated with cortical bone microstructural alterations and bone fragility in db/db male mice

doi: 10.3389/fcell.2025.1562764

Figure Lengend Snippet: Effects of high glucose and recombinant Sclerostin (SCL) on osteocyte remodeling markers in IDG-SW3 cells. (A) Representative images showing osteocytic differentiation over 28 days, including DMP1-GFP fluorescence, ALP staining, and ARS staining. (B) Time-course expression of late osteocytic markers (FGF23, MEPE, Pdpn, Phex, and Sost) over 28 days, presented as relative fold induction. (C) Elevated mRNA expression of Sost, Ctsk, Mmp-13, and Atp6v0d2 in IDG-SW3 cells treated with 20 mM glucose compared to control cells. (D) Upregulation of Ctsk and Mmp-13 mRNA levels in IDG-SW3 cells treated with recombinant SCL compared to vehicle control. Data represent mean ± SD from three independent experiments (n = 3). Statistical significance is indicated (*p < 0.05, **p < 0.01, ****p < 0.0001).

Article Snippet: For recombinant sclerostin (SCL) treatment, IDG-SW3 cells were treated with 100 ng/mL recombinant mouse sclerostin (HY- P70717 , MCE, China) for 48 h following 28 days of osteogenic induction.

Techniques: Recombinant, Fluorescence, Staining, Expressing, Control

Journal: JCI Insight

Article Title: Activation of connexin hemichannels enhances mechanosensitivity and anabolism in disused and aged bone

doi: 10.1172/jci.insight.177557

Figure Lengend Snippet: After a 4-week HLS in 16-week-old male mice, PGE 2 levels and SOST were assessed. ( A and B ) ELISA analysis of PGE 2 level in bone marrow–flushed femur diaphysis ( A ) and hindlimb bone marrow for both control and HLS tibias of vehicle- and Cx43(M2)-treated mice ( B ). ( C and D ) Representative COX-2 immunohistostaining and quantification of COX-2 + osteocytes (black arrows) in middiaphyseal cortical bone. Scale bar: 40 μm. n = 5–6 per group. ( E and F ) Representative SOST immunohistostaining and quantification of SOST + osteocytes (black arrows) in middiaphyseal cortical bone. Scale bar: 40 μm. n = 5–6 per group. Black and red arrowheads indicate positive and negative osteocytes, respectively. Data are expressed as mean ± SD. * P < 0.05; ** P < 0.01. Statistical analysis was performed using 2-way ANOVA with Tukey test for differences among groups ( A , B , D , and F ).

Article Snippet: Briefly, after antigen retrieval, described previously , sections were probed with primary antibodies against RANKL (ab9957, 1:200, Abcam), COX-2 (12375-1-AP, 1:200, Proteintech), SOST (AF1589, 1:400, R&D Systems), and unphosphorylated β-catenin (ab16051, 1:200, Abcam), followed by counterstaining with hematoxylin.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: JCI Insight

Article Title: Activation of connexin hemichannels enhances mechanosensitivity and anabolism in disused and aged bone

doi: 10.1172/jci.insight.177557

Figure Lengend Snippet: ( A and B ) ELISA analysis of PGE 2 level in bone marrow–flushed tibial diaphysis ( A ) and serum after 5 days of mechanical loading ( B ). n = 6 per group. IHC was performed on diaphyseal 37% cortical bone after 5 days/week loading for 2 weeks. ( C and D ) Representative COX-2 immunohistostaining and quantification of COX-2 + osteocytes in diaphyseal 37% cortical bone. Scale bar: 40 μm. n = 5 per group. ( E and F ) Representative SOST immunohistostaining and quantification of SOST + osteocytes in diaphyseal 37% cortical bone. Scale bar: 40 μm. n = 5 per group. ( G and H ) Representative β-catenin immunohistostaining and quantification of β-catenin + osteoblasts on the endosteal surface of diaphyseal 37% cortical bone. Scale bar: 50 μm. n = 5 per group. Black arrowheads indicate positive osteocytes. Data are expressed as mean ± SD. * P < 0.05; ** P < 0.01. Statistical analysis was performed using the paired Student’s t test for loaded and contralateral tibias ( A , D , F , and H ), unpaired Student’s t test for the serum PGE 2 level ( B ), and 2-way ANOVA with Tukey test for differences among groups ( A , D , F , and H ). Ps, periosteal surface; Ec, endosteal surface.

Article Snippet: Briefly, after antigen retrieval, described previously , sections were probed with primary antibodies against RANKL (ab9957, 1:200, Abcam), COX-2 (12375-1-AP, 1:200, Proteintech), SOST (AF1589, 1:400, R&D Systems), and unphosphorylated β-catenin (ab16051, 1:200, Abcam), followed by counterstaining with hematoxylin.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Activation of connexin hemichannels enhances mechanosensitivity and anabolism in disused and aged bone

doi: 10.1172/jci.insight.177557

Figure Lengend Snippet: Enhanced Cx43 HC activity releases PGE 2 , leading to a reduction of SOST expression in osteocytes during both mechanical loading and unloading. Aging is associated with a decline in osteocytic Cx43 levels and reduced responsiveness of Cx43 HCs to mechanical loading. Activation of osteocytic Cx43 HCs using the Cx43(M2) antibody increases PGE 2 levels and suppresses SOST expression, resulting in increased endosteal osteoblast activity and bone formation, while simultaneously decreasing endosteal osteoclast activity in aged mice under mechanical loading conditions. During periods of disuse, apoptotic osteocytes release higher levels of RANKL, which induces osteoclast differentiation and recruitment. Activating HCs with the Cx43(M2) antibody raises extracellular PGE 2 levels. This increase in PGE 2 suppresses SOST, thereby enhancing endosteal bone formation and preventing osteocyte apoptosis and RANKL expression in osteocytes. Consequently, reduced endosteal osteoclast activity leads to decreased bone resorption.

Article Snippet: Briefly, after antigen retrieval, described previously , sections were probed with primary antibodies against RANKL (ab9957, 1:200, Abcam), COX-2 (12375-1-AP, 1:200, Proteintech), SOST (AF1589, 1:400, R&D Systems), and unphosphorylated β-catenin (ab16051, 1:200, Abcam), followed by counterstaining with hematoxylin.

Techniques: Activity Assay, Expressing, Activation Assay