Hypoxia induction and caffeine treatment

For hypoxic experiments, the cell monolayers in culture dishes (6 × 10⁵ cells/dish) were transferred into a hypoxic chamber (Stemcell Technologies Inc., Vancouver, Canada) containing premixed gas (1% O₂, 5% CO₂, and 94% N₂) for 12 h. The temperature inside the chamber was set at 37°C, and the chamber was moistened with a dish containing sterile water. In parallel, the cells cultured in a humidified incubator with 21% O₂ and 5% CO₂ at 37°C served as the control (normoxia). To evaluate the effect of caffeine during hypoxia, the cells cultured under hypoxic condition were co-treated with various doses (3.125, 6.25, and 12.5 mM) of caffeine (Sigma–Aldrich).