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.