HIF-1α Stabilization and Function
The resultant effect on PHD2 activity was determined using a
fluorescence-based assay (McNeill et al. , 2005) and the HIF-1α
556–574 peptide substrate (containing the PHD2 hydroxylation site;
19-mer fragment; AnaSpec) in cytoplasmic extracts. Resultant effect on
HIF-1α stabilization was then determined by stain-free,
semi-quantitative Western blotting (WB) (Zhu et al. , 2009).
Briefly, protein extracts were separated using stain-free, tris/glycine
12% polyacrylamide gels and imaged before and after transfer to
nitrocellulose and blotting (ChemiDoc™; Bio-Rad) to determine expressed
protein levels relative to the total loaded protein in the gel.
Expression of selected gene targets of HIF-1α and chondrogenesis (Table
S2) were determined by real-time quantitative PCR (qPCR) (Brenneret al. , 2014). Briefly, harvested constructs were snap frozen
(liquid N2), and total RNA was extracted (RNeasy Mini
Kit; Qiagen) and reversed transcribed (SuperScript III Reverse
Transcriptase; Invitrogen). qPCR was performed using an ABI ViiA PCR
system (ThermoFisher) with the Power SYBR Green PCR Master Mix (Applied
Biosystems) and 18S (ribosomal RNA) as the endogenous (internal) control
(Brenner et al. , 2014). PCR primers were constructed using
Primer-BLAST (NCBI) and published mRNA sequences (NIH GenBank).
Expression levels were calculated using the ΔΔCT method.
Loss-of-function studies were also conducted using YC-1 to degrade
HIF-1α (Chun et al. , 2001) with the resultant effect on
extracellular matrix synthesis determined by radioisotope incorporation
(Waldman et al. , 2003). After 4 weeks of culture, samples were
incubated in YC-1 (10 µM) for 48 hours in the presence of
[3H]-proline (to quantify synthesis of collagen)
and [35S]-sulfate (to quantify synthesis of
proteoglycans) (2.5 µCi each isotope; Perkin Elmer). After incubation,
cultures were washed to remove unincorporated isotope and digested in
papain (refer to Biochemical Quantification of Accumulated
Tissue ) with aliquots used for β-liquid scintillation counting (Beckman
Coulter). Isotope incorporation was expressed relative to DNA content
(refer to Biochemical Quantification of Accumulated Tissue ) and
expressed relative to the controls.