The hypoxia-inducible factor α pathway couples angiogenesis to osteogenesis during skeletal development
J. Clin. Invest. Ying Wang, et al. 117:1616 doi:10.1172/JCI31581 [
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Figure 6Deletion of
Vhl in primary osteoblasts in vitro does not affect osteoblast proliferation and apoptosis.
Confluent
Vhl floxed primary osteoblast monolayers were infected with either adeno-GFP or adeno-CreM1 (100 MOI).
Vhl mRNA expression in infected osteoblasts was determined by real-time PCR 48 hours after infection to assess deletion efficiency. Cell proliferation, apoptosis, and differentiation assays were performed as described in Methods. (
A and
B) Cell proliferation was assessed by flow cytometry using BrdU incorporation. (
C and
D) Cell apoptosis was assessed by flow cytometry using annexin V–PE staining. (
E) Mineralized nodule formation was determined by ALP (left) and von Kossa staining (right) 7 and 14 days after cells were cultured in osteogenic medium. (
F and
G) Densitometric analysis of ALP and von Kossa staining observed in
E using NIH ImageJ 1.36b. Data represent mean ± SEM. (
H) Measurement of
Vhl,
Hif1a, runt-related transcription factor 2 (
Runx2), and
OC mRNA expression by quantitative real-time PCR at day 14 of osteogenic induction. **
P < 0.01; ***
P < 0.001.
