Fig. 2

ApoE impairs osteoblast differentiation and inhibits Wnt/β-catenin and Hippo signaling. a Schematic diagram of BMSC isolation, culture, subsequent osteogenic differentiation with vehicle or rApoE treatment and subsequent analysis. b BMSCs from 24-month-old C57BL/6 mice were cultured and differentiated in osteogenic media containing vehicle or rApoE. Wells were washed, fixed, and stained for Alkaline Phosphatase (Alk. Phos.) or mineral (Alizarin Red) to assess osteoblast differentiation and matrix mineralization, respectively. c–f Transcripts for osteogenic genes (Alp, Bsp, Col1, and Ocn) were measured using RT-PCR (n = 6). g Bulk RNA-seq was used to identify differentially expressed genes (DEGs) which were presented on a volcano plot using a log₂(FC)-value of 2 and a P value of 0 as cut-offs for DEGs to compare vehicle-treated vs. rApoE-treated groups. h Downregulated DEGs were collected for Gene Ontology (GO) biological process analysis and (i) KEGG pathway analysis. j Western blot analysis of active-\({\rm{\beta }}\)-catenin and total \({\rm{\beta }}\)-catenin protein levels in vehicle-treated and rApoE-treated groups. k Immunofluorescence staining (active-\({\rm{\beta }}\)-catenin) of vehicle- and rApoE-treated osteogenic cultures. l, m Expression level of\(\,{\rm{\beta }}\)-catenin target genes Axin2 and Cyclin D1 (n = 6). n Western blot analysis of active-Yap and total-Yap in vehicle-treated and rApoE-treated groups. o Immunofluorescence staining (active-Yap) of vehicle- and rApoE-treated osteogenic cultures. p–r Expression level Yap target genes Axl, Ctgf, and Cyr61 (n = 6). Data are presented as mean ± 95% confidence interval. *P < 0.05