Journal: Molecular Medicine

Article Title: Erythropoietin regulates osteoclast formation via up-regulating PPARγ expression

doi: 10.1186/s10020-024-00931-7

Figure Lengend Snippet: EPO enhances RANKL-induced osteoclast differentiation and bone resorption in vitro. (A) Representative TRAP staining images of BMMs treated with 30 ng/mL M-CSF and 50 ng/mL RANKL for four days in the presence of EPO and EMP9. Scale bar = 200 μm. (B) Representative images of osteoclasts with actin ring (red) and cell nucleus (blue); the BMMs were treated with 30 ng/mL M-CSF and 50 ng/mL RANKL for four days in the presence of EPO or/and EMP9. Scale bar = 200 μm. (C) Representative images of bone resorption pits on Corning Osteoassay 24-well plates. Scale bar = 200 μm. (D) The area of TRAP + multinucleated cells (nuclei > 3) was quantified in each group. (E) The relative cell size of TRAP + multinuclear cells (nuclei > 3) was quantified in each group. (F) EPO increased the number of multinucleated cells (nuclei > 3) with actin rings. (G) The relative resorption area was quantified by using ImageJ Software. Error bars are mean ± SD of triplicate experiments; * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant

Article Snippet: Scale bar = 200 μm. (C) Representative images of bone resorption pits on Corning Osteoassay 24-well plates.

Techniques: In Vitro, Staining, Software

Journal: Molecular Medicine

Article Title: Erythropoietin regulates osteoclast formation via up-regulating PPARγ expression

doi: 10.1186/s10020-024-00931-7

Figure Lengend Snippet: EPO binding to its receptor EPOR activates the Jak2/ERK signal pathway, increasing PPARγ expression. Then, PPARγ directly regulated the expression of c-fos to promote osteoclast differentiation and enhanced osteoclastic bone resorption

Article Snippet: Scale bar = 200 μm. (C) Representative images of bone resorption pits on Corning Osteoassay 24-well plates.

Techniques: Binding Assay, Expressing

Journal: Nutrients

Article Title: Cordycepin Prevents Bone Loss through Inhibiting Osteoclastogenesis by Scavenging ROS Generation

doi: 10.3390/nu8040231

Figure Lengend Snippet: Cordycepin reduces osteoclastic bone resorption activity in a dose-dependent way. ( A ) Representative images of primary BMMs cultured on bovine slices treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for five days with different concentrations of cordycepin treatment (0 μg/mL, 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, and 5 μg/mL). ( B ) Quantification of RANKL induced osteoclastic bone resorption in the bone slices group. ( C ) Representative images of Osteoassay surface 96-well plate after removal of osteoclasts. ( D ) Quantification of RANKL induced osteoclastic bone resorption in the osteo surface group. Scale bar represents 400 μm. Data in the figures represent average ± SD. ** p < 0.01, *** p < 0.001 based on one way ANOVA.

Article Snippet: Osteo Assay Surface for bone resorption was purchased from Corning (Corning, NY, USA).

Techniques: Activity Assay, Cell Culture

Journal: International Journal of Biological Sciences

Article Title: Knockout and Double Knockout of Cathepsin K and Mmp9 reveals a novel function of Cathepsin K as a regulator of osteoclast gene expression and bone homeostasis

doi: 10.7150/ijbs.72211

Figure Lengend Snippet: The double deletion of Ctsk and Mmp9 impairs osteoclast function in vitro . ( a ) TRAP staining shows decreased OC formation in MBM cells isolated from Mmp9 -/- ,Ctsk -/- and Ctsk -/- / Mmp9 -/- cells that were stimulated by M-CSF and RANKL for 5 days. Higher magnification images are shown in the lower panels. (b) MBM cells isolated from WT, Ctsk -/- , Mmp9 -/- , or Ctsk -/- / Mmp9 -/- mice and then treated with M-CSF and RANKL for 5 days to promote OC differentiation. (b, Top panel) Acridine orange staining revealed that extracellular acidity (red-orange) was significantly reduced in DKO cells. (b, Bottom panel) F-actin ring formation assay for cell cultures shows disrupted or absent ringed structures of F-actin dots (actin rings) in Ctsk -/- /Mmp9 -/- and Ctsk -/- mice. Inset is the magnified image of the boxed areas. (c) Quantification for panels A and B (n=5 independent experiments). (d) Pre-OC seeded on bone slices were cultured with M-CSF and RANKL for 6 days before being submitted to bone resorption analysis. WGA-FITC staining revealed a drastic decreased in bone resorption in Ctsk -/- OCs and a near absence of bone resorption in Ctsk -/- Mmp9 -/- OCs, but Mmp9 -/- and WT cells showed similar bone resorption abilitie©( e ) Quantification for D is shown (n>20, Pit depth). All the data are presented as mean values ± SD; two-tailed unpaired t test; ns. non-significant.

Article Snippet: ELISA kits used were CTX/C-telopeptide assay for bone resorption (MyBioSource, cat# MBS724196), P1NP/total procollagen type 1 N-terminal propeptide (R&D Systems, cat# DY6220-05).

Techniques: In Vitro, Staining, Isolation, Tube Formation Assay, Cell Culture, Two Tailed Test