FIGURE 4:
Hypoxia-induced Nodal expression persists following up to 48 h of reoxygenation. (A to C) Immunoblot analyses of Nodal protein in (A) MCF-7, (B) T47D, and (C) C81-61 cells cultured in 20% O2 (24 h), 0.5% O2 (24 h), or 0.5% O2 (24 h), which was followed by reoxygenation in 20% O2 (24–72 h). Results indicate that hypoxia-induced Nodal expression is still elevated after up to 48 h of reoxygenation compared with 20% O2 controls. The pro-Nodal (∼39 kDa) band is presented, and β-actin is used as a loading control. (D and E) Real-time RT-PCR analyses of Nodal mRNA in (D) T47D and (E) C81-61 cells cultured in 20% O2, 0.5% O2, or 0.5% O2, which was followed by reoxygenation in 20% O2 (24–72 h). Nodal mRNA underwent an initial drop in hypoxia (24 h), which was followed by a significant up-regulation during reoxygenation. Gene expression was normalized to RPLPO, and bars represent mean gene expression ± SD relative to 20% O2 control. Different letters are significantly different (n = 6; p < 0.05).