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: 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

Journal: Journal of Bone Metabolism

Article Title: Inhibitory Effect of Rosae Multiflorae Fructus Extracts on the Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis through Modulation of P38- and Ca 2+ -Mediated Nuclear Factor of Activated T-Cells Cytoplasmic 1 Expression

doi: 10.11005/jbm.2020.27.1.53

Figure Lengend Snippet: Effect of Rosae Multiflorae fructus extract (RMF-E) on osteoclast differentiation and resorption activity. Bone marrow-derived macrophages were cultured with the indicated concentrations of RMF-E, under receptor activator of nuclear factor-κB ligand (100 ng/mL) and macrophage-colony stimulating factor (30 ng/mL) treatment, for 4 days. (A) Osteoclasts were fixed and stained for tartrate-resistant acid phosphatase (TRAP). (B) TRAP + -multinuclear cells with ≥3 nuclei were counted as mature osteoclasts. (C) Total TRAP activity from TRAP + -mono-, di-, and multi-nuclear cells were measured at an absorbance of 405 nm (A 405 nm ). (D) F-actin rings were stained with rhodamine-phalloidin in osteoclasts treated with/without RMF-E (20 µg/mL). (E) The resorption activity of osteoclasts treated with/without RMF-E (20 µg/mL) was measured by pit formation on hydroxyapatite-coated plates, after 7 days. Data are expressed as the mean±standard deviation and are representative of 3 independent experiments. * P <0.05, ** P <0.01, and *** P <0.001 vs. the non-treated control (NC, 0 µg/mL RMF-E) (Scale bar=200 µm). NS, not significant.

Article Snippet: To measure bone resorption activity, BMMs were cultured in a Cosmo Bio 48-well bone resorption assay plate (Oriental Yeast Co., Ltd., Tokyo, Japan) under M-CSF and RANKL treatment with/without RMF-E (20 μg/mL) for 7 days, and then cleared using bleach.

Techniques: Activity Assay, Derivative Assay, Cell Culture, Staining, Standard Deviation, Control