Effects of hypoxia or CoCl2 treatment on HIF-1 DNA binding and reporter gene activity in astrocytes. (A) Mouse HIF-1α+/+ and HIF-1α+/- cells were exposed to hypoxia or 125 μM CoCl2 for 6 h, nuclear extracts were then prepared and EMSA carried out as described in the Materials and Methods. HIF-1 in the nuclear extracts isolated from hypoxia or CoCl2-treated cells bound to the wild-type probe (lanes #1–6) but not to the mutant probe (lanes #7–12). HIF-1/DNA complex was detected in hypoxia (H)-or CoCl2 (Co)-treated cells (lanes #2, 3, 5, 6) but not in control (C) cells (lanes #1, 4). More complex (darker band) was seen in hypoxia-or CoCl2-treated HIF-1α+/+ cells (lanes #2, 3) than that in hypoxia-or CoCl2-treated HIF-1α+/- cells (lanes #5, 6). Supershift assay showed that the HIF-1/DNA complex was shifted up in the presence of wild-type (wt) oligo probe and 4 μg HIF-1α antiboby (lanes #13 & 14). (B) The activity of the reporter gene, luciferase yellow, under a wild-type HIF-1-binding sequence from MCP-1 promoter (pGL3/MCP1w) or a mutated sequence (pGL3/MCP1m), was carried out to test the transcriptional activation of MCP-1 by HIF-1 activated by hypoxia. FHAs were transfected with an empty vector, pGL3/MCP1w or pGL3/MCP1m, respectively, and recovered overnight for 16 h. The cells were exposed to normoxia or hypoxia for 4 h and then harvested for luciferase yellow assays. Hypoxia strongly stimulated the reporter gene activity from pGL3/MCP1w but not from the empty vector and pGL3/MCP1m. Each bar represents the mean ± SD of three assays and each assay had at least two replicates. Asterisks indicate significant difference compared to relevant controls (p < 0.05, one-way ANOVA, followed by multiple comparisons among means).