Figure 5. ROS generation is required for hypoxia-induced activation of PI3K, ERK and Rac1 and HIF-1α expression.

(A) Representative micrographs of MCF-7 cells growing under normoxia or hypoxia without or with 2 mM NAC and stained with CM₂-DCFHDA. (B) Effect of NAC on HIF-1α and VEGF mRNA expressions in hypoxic cells. After pretreatment with 2 mM NAC for 1 h, the cells were incubated under hypoxia for 1 h and HIF-1α and VEGF mRNA expressions was examined. (C&D) Effect of ROS inhibitor on hypoxia-stimulated PI3K, ERK and Rac1 activation and HIF-1α expression. After preincubation for 1 h with 2 mM NAC, cells were grown under hypoxia for 1 h and then cells were analyzed by Akt or ERK or Rac1 or HIF-1α assay as described in ‘Materials and methods’. Data are presented as mean ± SD of 5 independent determinations. *: P<0.05, referring to the difference between cells treated with and those without hypoxia. ^#: P<0.05 (t-test), refers to the comparison of cells treated with hypoxia plus NAC and the cells treated with hypoxia alone.