Journal: Bone Research

Article Title: Estradiol regulates osteoclast sialylation via ST3Gal1 in postmenopausal osteoporosis

doi: 10.1038/s41413-025-00498-x

Figure Lengend Snippet: RANKL induces St3gal1 expression is required for osteoclast formation. a Schematic of the differentiation process of bone marrow macrophages (BMMs) from wild-type C57BL/6 mice into pre-osteoclasts (pOC) and mature osteoclasts (mOC) under M-CSF and RANKL stimulation over a 5-day period. TRAP staining shows osteoclast formation at different stages. Scale bar = 100 μm. b Heatmap showing the expression levels of the mouse sialyltransferase (ST) gene family during osteoclast differentiation from BMMs to pOCs and mOCs. c ATAC-seq data illustrating chromatin accessibility changes at the loci of ST3Gal1 and ST6Gal family members during osteoclast differentiation. d Immunofluorescence staining of α2,3 and α2,6 sialylation on BMMs after treatment with M-CSF and/or RANKL. Scale bar = 20 μm. e Quantification of mean fluorescence intensity of α2,3 and α2,6 sialic acids on BMMs treated with M-CSF and RANKL ( n = 3). f TRAP staining of osteoclast cultures treated with vehicle or sialidase. Scale bar = 100 μm. Quantification of the number of multinucleated osteoclasts per well in vehicle- and sialidase-treated cells ( n = 3). g TRAP staining of osteoclasts treated with vehicle or siRNA targeting ST3Gal1 (si-ST3Gal1). Scale bar = 100 μm. Quantification of the number of multinucleated osteoclasts per well in vehicle- and si-ST3Gal1-treated cells ( n = 3). Data are presented as mean ± SD; statistical significance was determined by one-way ANOVA, with * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: SialEXO 23 α2-3 specific sialidase (5 units, Genovis Inc.) was preincubated in 20 mmol/L Tris (pH 7.5, 37 °C, 1 h) and injected intrafemorally.

Techniques: Expressing, Staining, Immunofluorescence, Fluorescence

Journal: Bone Research

Article Title: Estradiol regulates osteoclast sialylation via ST3Gal1 in postmenopausal osteoporosis

doi: 10.1038/s41413-025-00498-x

Figure Lengend Snippet: Bone sialylation level and ST3Gal1 expression are increased in ovariectomized mouse. a Representative immunofluorescent images of femur sections from sham and OVX mice showing α2,3-sialylation (green) and TRAP + osteoclasts (red). The growth plate (GP), metaphysis, and trabecular bone (TB) regions are indicated. Scale bars = 100 μm. b Quantification of TRAP + area and α2,3-sialic acid (SA) + area as a percentage of the total bone area in sham and OVX mice ( n = 5). c Representative immunohistochemical (IHC) images of ST3Gal1 expression in femur sections from sham and OVX mice. Insets show magnified views of the indicated regions. Scale bars = 200 μm (top panels), 100 μm (bottom panels). d Quantification of ST3Gal1 + area as a percentage of the total bone area in femur sections from sham and OVX mice ( n = 5). e Line plot of relative fluorescence intensity profiles of TRAP and α(2,3)-SA signals along selected regions in ( a ), demonstrating colocalization. f Quantification of α(2,3)-SA⁺ osteoclasts (TRAP⁺) as a percentage of total osteoclasts in sham and OVX mice ( n = 3). Data are presented as mean ± SD; statistical significance was determined by one-way ANOVA, with * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: SialEXO 23 α2-3 specific sialidase (5 units, Genovis Inc.) was preincubated in 20 mmol/L Tris (pH 7.5, 37 °C, 1 h) and injected intrafemorally.

Techniques: Expressing, Immunohistochemical staining, Fluorescence

Journal: Bone Research

Article Title: Estradiol regulates osteoclast sialylation via ST3Gal1 in postmenopausal osteoporosis

doi: 10.1038/s41413-025-00498-x

Figure Lengend Snippet: Estradiol inhibits RANKL-activated St3gal1 transcription via competitively binding with TRAF6, reducing c-Fos activity. a TRAP staining of osteoclasts cultured with M-CSF, RANKL, or RANKL + estradiol (E2) for 48 and 120 h. Scale bars = 100 μm. b Quantification of osteoclasts per well in the conditions described in ( a ) at 48 and 120 h. qPCR analysis of St3gal1 mRNA levels in osteoclasts treated with M-CSF, RANKL, or RANKL + E2 for 48 h ( c ) and 120 h ( d ). e Immunofluorescent staining of α2,3 sialylation (red) in osteoclasts treated with M-CSF, RANKL, or RANKL + E2 at 48 and 120 h. DAPI (blue) is used to counterstain nuclei. Scale bar = 20 μm. f Quantification of mean fluorescence intensity of α2,3 sialic acid staining in osteoclasts treated as described in ( e ). g Co-immunoprecipitation (IP) of ERα and TRAF6 in osteoclasts treated with RANKL or RANKL + E2, followed by immunoblotting (IB) to detect TRAF6, ERα, and c-FOS. h Immunofluorescence analysis of p65 nuclear translocation in osteoclasts treated with RANKL or RANKL + E2. DAPI (blue) counterstains nuclei, and Phalloidin (green) stains actin. Scale bar = 20 μm. i Quantification of p65 nuclear translocation (percent of positive nuclei and mean intensity) in osteoclasts treated with RANKL or RANKL + E2 ( n = 5). Data are presented as mean ± SD; statistical significance was determined by one-way ANOVA, with * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: SialEXO 23 α2-3 specific sialidase (5 units, Genovis Inc.) was preincubated in 20 mmol/L Tris (pH 7.5, 37 °C, 1 h) and injected intrafemorally.

Techniques: Binding Assay, Activity Assay, Staining, Cell Culture, Fluorescence, Immunoprecipitation, Western Blot, Immunofluorescence, Translocation Assay