Hypoxia Mimetic and HIF-2α Knockdown Enhance Temozolomide Sensitivity

For experiments mimicking hypoxic conditions, cells were cultured with 100 μM deferoxamine mesylate (DFX) (Sigma-Aldrich), a hypoxia mimetic^{[21 (link),22 (link)]}. A Silencer Select short interfering RNA (siRNA) against human HIF-2a (Dharmacon, Lafayette, CO) was delivered to cells using two different transfection systems: (1) Lipofectamine RNAiMAX as described by the manufacturer (Invitrogen); and (2) cationic amphiphilic micelles complexed with transfection lipids as described in a previous publication^{[23 (link)]}. Silencer Select Negative Control siRNA (Invitrogen) or Luciferase siRNA (Invitrogen) was used as a control, nontargeting sequence. After 24 h, 1 mM temozolomide (Invitrogen) was added to the media. After 48 h, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Promega, Madison, WI) was performed to evaluate viability as a measure of chemotherapeutic response to a combined HIF-2a siRNA and temozolomide therapy.