Effect of purine nucleosides on HIF-1α protein accumulation, and DNA-binding capacity in PC12 cells: PC12 cells were exposed to normoxia and hypoxia (1% oxygen) for 24 hours in serum-reduced medium. Prior to hypoxic insult cells were treated without purine nucleosides (Co) or with 500μM adenosine (Ado) and inosine (Ino). HIF-1α protein levels of (A) total cell extracts and (B) nuclear extracts were determined by western blot analysis. DNA polymerase was used as a loading control and the ratios of HIF-1α/DNA polymerase are shown for normoxia (grey) and hypoxia (black) in control cells (Co) in panels Aa and Ba. Nuclear extracts were also treated with CoCl2 (positive control). Levels of hypoxic HIF-1α were the basis for calculation of the additional purine-mediated increase (Ab and Bb). (C) Nuclear accumulation of HIF-1α was tested by immunofluorescence analysis. (Panels a-e) Hoechst stain (blue) was used to visualize the nucleus of the cells; (Panels f-j). An FITC-labeled secondary antibody (green) was used to visualize HIF-1α, and (panels k-o) the same cells are shown under phase contrast optics. (C Panels a, f and k) CoCl2-treated samples are shown for comparison. (D) The DNA-binding capacity of nuclear extracts was analyzed by EMSA kit. The colorimetric readout was quantified by spectrophotometry. Fold increases over control (Co) indicate increases in nuclear HIF-1α binding to immobilized DNA. Values represent the mean ± S.E.M., n=3-9. Differences were analyzed using unpaired one-tailed t-test: (A) **p<0.01, ***p<0.001, (B) *p<0.05. Scale bar: (C) 20μm.