Figure 2.

Hypoxia-induced increase in VEGF expression is ERK-dependent but PI3K-independent in glioblastoma cells. (A/B/C) SF-268 cells were subjected to hypoxia using cobalt(II) chloride hexahydrate (CoCl₂) for the indicated time. Cells were then lysed, and the lysates were blotted for p-ERK and ERK (A) and p-Akt and Akt (B), as well as PIP₃ and β-actin for loading control (C). The graphs in each panel are densitometric analysis of the Western blots using Image J. Values are normalized to the loading control (ERK, Akt, and β-actin for p-ERK, p-Akt, and PIP₃, respectively) and expressed as fold change compared to time zero (normoxia). (D/E) SF-268 cells were treated with 50 μM U0126 (with DMSO as a carrier) for 24 h (D) or with wortmannin 100 nM (Wm) (with DMSO as a carrier) for 4 h (E) or with DMSO as a control. Cells were then subjected to 4 h hypoxia and lysed, and cell lysates were blotted for VEGF-A or β-actin for loading control. The graphs are quantitations for the VEGF bands in (D/E) normalized to actin and expressed as fold change compared to control (DMSO). (F) U87 cells were treated with 50 μM U0126 for 24 h or with wortmannin 100 nM (Wm) for 4 h (with DMSO as a carrier). Cells were then subjected to 4 h hypoxia and lysed, and cell lysates were blotted for VEGF-A or β-actin for loading control. The graphs are quantitations for the VEGF bands in (F) normalized to actin and expressed as fold change compared to control (DMSO). (G) ELISA for supernatants from SF-268 cells (upper graph) or U87 cells (lower graph), treated with U0126 or wortmannin or DMSO alone and then kept in normoxia or subjected to 4 h hypoxia. Supernatants were collected and measured for VEGF-A secretion according to the manufacturer’s guidelines. Values are expressed as fold change at every treatment to normoxia. (H) (+ indicates addition of the treatment) SF-268 cells were treated with DMSO or with 50 μM U0126 for 24 h, subjected to hypoxia for 4 h. Cells were then lysed, and the lysates were blotted for p-Akt and Akt. The graph is a quantitation of the gels. Values are normalized to Akt and expressed as fold change compared to control (normoxia + DMSO alone). The data are means ± SEM from three different experiments; * p < 0.05 indicates statistically significant differences.