Figure 3.

Amphioxus HIFα had a functional ODD domain, and different HIFα isoforms had different responses to hypoxia. A) Effects of hypoxia and the deletion of ODD domain. HEK 293T cells transfected with the indicated amphioxus HIFα constructs or empty vector (control) were subjected to 24 h hypoxia (1% oxygen) or kept under normoxia (ambient air). Cells were lysed and analyzed by Western immunoblot with the indicated antibodies. Asterisks indicate nonspecific bands. B) Effect of CoCl₂. Cells transfected with pCS2-EGFP-amHIFaIa or mock-transfected cells were treated with 50 μM CoCl₂ for 24 h and analyzed as described above. C) Amphioxus HIFα ODD domain conferred oxygen responsiveness. HEK 293T cells were transfected with pBIND-ODD-ARNT together with pG5luc. Gal4-AmHIF-ODD-ARNT contains the amphioxus HIFα ODD domain fused to the TAD of human ARNT. At 1 d after transfection, cells were switched to hypoxia or kept in normoxia for 24 h and lysed, and luciferase activity was measured. Transactivation activity is expressed as fold increase over the normoxia group. Values are means ± sd (n=3). *P < 0.05. D) Effects of hypoxia on the amphioxus HIFα isoforms. Cells transfected with pCS2-EGFP-amHIFαIa, Ib, Ic, or Id were subjected to 24 h hypoxia (1% oxygen) or normoxia (ambient air) and analyzed by Western immunoblot with the indicated antibodies. Empty transfected cells were used as the control.