Figure 2.

Effect of chemical hypoxia on HIF-1α expression and ROS generation on SH-SY5Y. (A) Protein expression levels of HIF-1α were detected by Western blot in SH-SY5Y cells in induced hypoxia with CoCl₂ (0, 50, 100, or 200 μM) for 24 h. α-tubulin levels are presented as a loading control. (B) Relative levels of HIF-1α protein expression following treatment with CoCl₂. Data are presented as the mean ± standard deviation of three independent experiments, and results normalized to untreated control cells. *** p < 0.001 vs. the unexposed control cells according to Student’s t-tests. (C) Flow cytometric analysis of cytosolic ROS generation using DCFH-DA and mitochondrial superoxide radicals using MitoSOX in SH-SY5Y cells treated with CoCl₂ (0, 50, 100, or 200 μM) for 24 h. (D) Representative fluorescent images of DCFH-DA (green) staining and MitoSOX (red) staining in SH-SY5Y cells treated with 200 μM CoCl₂ were obtained using confocal microscopy. Non-treated SH-SY5Y cells were used as negative controls. ×200 magnification.