Figure 5.

Effects of copper sulfate on expression of HIF-1α in normoxic and hypoxic HeLa cells. (A,B) HeLa cells were incubated for 6 h with the indicated concentrations of copper sulfate under normoxia and hypoxia (1% O₂). (A) Cell lysates were subjected to western blot analysis using antibodies against HIF-1α, p53, DEC1, p21, total and phosphorylated (p)-ERK, and total and cPARP. (B) RT-PCR analysis for HIF-1α, VEGF, and DEC1. ACTN was the protein loading control; GAPDH mRNA was the mRNA loading control. (C) HeLa cells were incubated for 8 h with the indicated compounds (300 μM), after which cell lysates were subjected to western blot analysis using antibodies against HIF-1α, p53, DEC1, p21, total and p-ERK, and total and cleaved PARP. ACTN was the protein loading control. The results are representative of three independent experiments. Protein and PCR bands were quantified through pixel density scanning and evaluated using ImageJ, version 1.44a (http://imagej.nih.gov/ij/). The fold (shown above the bands) was normalized to the internal control protein (ACTN) or gene (GAPDH). Phosphorylated ERK after normalization to total ERK protein and cPARP fragment after normalization to full-length PARP protein are shown as fold changes.