Fig. 1.
Expression of a gain-of-function mutation of HIF2 in BMSCs inhibits osteoblastogenesis in vitro. a ALP staining and b Alizarin Red S staining of BMSCs isolated from HIF2dPAf/f mice, transduced with Ad-LacZ or Ad-Cre and cultured in osteogenic medium for 7 and 21 days, respectively. Representative images are shown on the left, and quantification of all biological replicates on the right. c, d qRT-PCR of total RNA extracted from the same BMSC cultures at d7 (c) and d21 (d). mRNAs encoding osteoblast markers (Runx2, Col1a1, Sp7, Alpl, Ibsp, Spp1, Ocn, Opg, Rankl, and Opg/Rankl ratio), and known downstream targets of HIF2 (Vegfa and Opg) are shown. Note that Ocn mRNA was virtually undetectable in both mutant and control cultures at d7, which is consistent with the early stage of osteoblastic differentiation of BMSCs at this time point. Data were normalized to expression of TATA-Box Binding Protein (TBP) mRNA. *P < 0.05