Figure 1.

Schematic representation of the experimental procedure. (A) The experiments can be divided into two separate parts. First, generation of a sub-cell line by treatment of PC12 cells with recurrent cycles of hypoxia (hypoxia–reoxygenation cycles), based on the hypothesis that this procedure would lead to the generation of a pseudohypoxic phenotype in these cells. In the second part, cells were characterized under normoxic conditions to confirm the pseudohypoxic phenotype of the newly established sub-cell line. (B) Procedure to generate PC12 sub-cell lines. Cells were treated with recurring cycles of hypoxia (≤1% oxygen, 24 h hypoxia/cycle, followed by a reoxygenation phase of 3–4 days). After 20 (PC12 Z20) cycles, morphological changes were determined by phase contrast microcopy in comparison with control cells cultivated under normoxic conditions (PC12 Z20 control). Furthermore, sub-cell lines after 10 hypoxia–reoxygenation cycles were obtained and characterized under normoxic conditions. Scale bar: 100 µm.