Siatf4

Cells were transfected with ON-TARGETplus SMARTpool siRNA against murine ATF4 (siATF4) or nontargeting control (siCtrl, Dharmacon, Lafayette, CO) using polyethylenimine (Polysciences, Warrington, PA) in serum-free media. After 12 h, transfection media was replaced with fresh serum-containing media, and cells were grown for an additional 48 h in hypoxia or normoxia prior to analysis.

The LN229, U87, T98G, HCT116, WC62, GBM12, and GBM22 cells were cultured in DMEM containing 10% FBS (Gemcell, New York, NY, USA), primocin (InvivoGen, San Diego, CA, USA), and in a humidified air (5% CO₂) at 37 °C. For the experiments, cells were cultured in DMEM containing 1.5% FBS (non-lipoprotein deficient) and primocin (InvivoGen). In selected instances, lipoprotein deficient serum was employed. NCH644, NCH421k, and NCH690 glioma stem-like cells were cultured in MG-43 medium (CLS, Heidelberg, Germany) for both maintenance and experiments [30 (link)]. A375 cells were cultured in RPMI medium 1640 containing 10% FBS (Gemcell), primocin (InvivoGen). For the experiments, cells were cultured in RPMI medium 1640 containing 1.5% FBS (non-lipoprotein deficient) and primocin (InvivoGen). Cells were transfected using either Lipofectamine RNAiMAX or Lipofectamine 3000 (Invitrogen) and harvested after 72 h after transfection. siTRAP1-2, siSREBP2, siPMAIP1 (siNoxa), and siBAK were purchased from Ambion. Non-targeting siRNA-pool (siNT), siTRAP1 (pool), and siATF4 were purchased from Dharmacon (Lafayette, CO, USA). siPERK-1 and siPERK-2 were purchased from Cell Signaling.