Figure 1.

Cytotoxic effects of low pO₂ levels on myeloma cell lines. (A) The % apoptosis (measured by cleaved caspase 3) following 72 hour culture of myeloma cells under “normoxic” conditions (~22% O₂, 5% CO₂) or hypoxic (0.1% O₂, 5% CO₂) conditions, except for OPM-2 cells which were cultured for 48 hours. Brackets indicate P<0.05 (T-test). Values represent mean ± 1 standard deviation of 4 independent experiments/cell line. (B). Immunoblots of HIF1α and HIF2α-subunit expression in 8226 and OPM-2 cells cultured under normoxic or hypoxic conditions (0.1% O₂, 5% CO₂) for 24 hours. The cells were harvested on ice then the cytoplasmic and nuclear fractions were isolated, and the lysate was immunoblotted for indicated proteins. C=cytoplasm fraction, N=nuclear fraction. The data presented are a representative immunoblots of at least 2 independent experiments. (C) Time course of CoCl₂-mediated induction of HIF1α expression in OPM-2 cells. Cells were cultured with 100nM CoCl₂ or vehicle control and the cellular lysates were harvested at indicated time points and immunoblotted for HIF1α expression. The data presented are a representative immunoblots of at least 2 independent experiments. (D) Hypoxia-mediated induction of HIF1α and HIF2α in MM1S, H929 and U266 myeloma cell lines cultured under normoxic or hypoxic (0.1% O₂, 5% CO₂) conditions for 24 hours. Cells were harvested on ice and the cell lysate was immunoblotted for HIF1α and HIF2α. The data presented are a representative immunoblots of at least 2 independent experiments. (E) Immunoblots showing hypoxia-mediated changes in survival-related protein expression in 8226 and OPM-2 cells cultured under normoxic or hypoxic conditions (0.1% O₂, 5% CO₂) for 24 hours. The cells were harvested on ice and the lysate was immunoblotted for indicated proteins. The data presented are a representative immunoblots of at least 2 independent experiments.