Journal: Bone Research

Article Title: Ubiquitin-specific protease 26 facilitates endochondral ossification by driving chondrocyte hypertrophy and mineralization

doi: 10.1038/s41413-026-00517-5

Figure Lengend Snippet: Compression loading induces the expression of USP26 through phosphorylation of estrogen receptor-α at serine 118. Dual-luciferase reporter assays to detect Usp26 promoter activity ( a ) and quantitative analysis of Usp26 mRNA expression ( b ) were conducted in chondrocytes under compression stimulation at levels of 0 and 15 kPa for a duration of 4 h, with or without ER-α knockdown. c The common binding sites of ER-α on the Usp26 gene promoter were further analyzed and screened through three public databases: hTFtarget, Jaspar, and HOCOMOCO. d The binding site mutation significantly impaired the Usp26 promoter activity induced by compression loading in primary human chondrocytes. e ChIP‒qPCR analysis of the binding sites of ER-α to the Usp26 gene promoter in primary human chondrocytes. f Western blot to detect the phosphorylation of serine 118 of ER-α under compression loading in primary human chondrocytes. The phosphorylation-deficient S118A mutation significantly attenuated the compression-induced increases in Usp26 promoter activity ( g ) and mRNA expression ( h ) in primary human chondrocyte. i The S118A mutation significantly restored the decreased protein level of FBP2 caused by compression loading. Immunofluorescence staining to detect phosphorylation of ER-α at S118 in chondrocytes during skeletal growth ( j ), bone fracture healing ( k ), and osteoarthritis ( l ). White and Red scale bars indicated 500 μm. m Schematic of USP26 facilitates endochondral ossification by driving chondrocyte hypertrophy and mineralization. *** P < 0.001. P -values were analyzed by two-way ANOVA in a , b , d , e and one-way ANOVA in g , h

Article Snippet: With 10 μL of ER-α antibody (CST, #13258) or negative control anti-IgG (CST, #2729), fragmented chromatin (200 μg) was incubated overnight to form immune complexes, which were coupled to Protein A beads.

Techniques: Expressing, Phospho-proteomics, Luciferase, Activity Assay, Knockdown, Binding Assay, Mutagenesis, Western Blot, Immunofluorescence, Staining