Figure 2. Hypoxia-induced CREB phosphorylation by induction of the MAPK/ERK signal transduction pathway.

A. The transcription of CREB and of hypoxic markers (GLUT1, VEGF) was analysed by qPCR. The bar charts represent the mean values and SEM of three independent experiments. B. CREB expression and phosphorylation was compared in HER-2/neu⁺ NIH3T3 cells under normoxia and hypoxia by Western blot analysis as described in Materials and Methods using an anti-CREB and anti-CREB phosphorylation-specific antibodies. One of three representative Western blots is shown. C. The activity/phosphorylation of the AKT and ERK pathway was determined under normoxic and hypoxic conditions by Western blot analysis using total and phosphorylation-specific antibodies, respectively. The data represent one of three biological replicates. D. Cells were treated under hypoxia with 5 μM LY294002, 100 nM trametinib or 5 μM U0126 for 24 h. The phosphorylation and total expression of CREB, AKT and ERK was analysed by Western Blot. E. Hypoxia-mediated induction of CREB phosphorylation and its dependence on the HER-2/neu status was determined in HTB122 cells and their HER-2/neu-transformed transfectants (E2A: dominant negative mutation in the HER-2/neu kinase domain; E2: wild-type HER-2/neu) either incubated under normoxia or hypoxia for 24 h, respectively, before Western blot analysis was performed as described in Materials and Methods. The results show one of two independent experiments (up). HTB122, E2 and E2A cells were incubated under normoxic conditions for 24 h before the HER-2/neu cell surface expression was determined using flow cytometry. The data are represented as histograms from one out of two representative experiments. The black area represents the IgG control, while the red defined area is the HER-2/neu-PE staining (down). F. Cells were incubated for 24 h under normoxia or hypoxia and the presentation of HER-2/neu on the cell surface was determined by flow cytometry as described in Materials and Methods using a PE-labelled anti-HER-/neu mAb. The bars represent the MFI of HER-2/neu compared to an IgG control from two independent experiments. G. The effect of inhibition of the HER-2/neu activity by treatment of parental HER-2/neu⁺ cells with increasing concentrations of lapatinib on CREB phosphorylation was analysed by Western blot. Cells were treated with lapatinib for 24 h under hypoxic conditions.