Response of ZF4 cells to hypoxic stress. (A) Cell Viability was analyzed with PrestoBlue™ HS Cell Viability Regent under normoxia or hypoxia treated for 1, 2, 3 and 4 d. (B) The mRNA expression of iron absorption and storage gene in ZFL cells was quantified by real-time RT–PCR under normoxia and hypoxia for 3 days. (C) Western blot analysis of Hif-1α and Ferritin expression in ZF4 cells cultured under normoxia and hypoxia for 3 days. (D) The microscope analysis of morphology changes in ZF4 cells under normoxia or hypoxia treated with or without FAC (2.5 mM), DFO (10 µM) and Fer-1 (2.5 µM) for 3 days. (E) Cell Viability was analyzed with PrestoBlue™ HS Cell Viability Regent under normoxia or hypoxia treated with or without FAC (2.5 mM), DFO (10 µM) and Fer-1 (2.5 µM). (F–H) Analysis of changes in general ROS (F), mitochondrial-derived ROS (G) and lipid peroxidation (H) levels in cells with H2DCFDA, MitoSOX and C11-BODIPY probe under normoxia and hypoxia for 3 days. Cells under hypoxic stress were rescued with FAC (2.5 mM), DFO (10 µM) and Fer-1 (2.5 µM). Normoxia was used as a control group for significance analysis. Error bars, mean ± s.d., n = 3 (biological replicates).
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