Panel A, clonogenic survival assay in U87MG cells after siRNA knockdown as indicated and treatment with increasing doses of DT-EGF, data represents the mean +/- SD of four experiments each performed in triplicate. Knockdown of autophagy regulators increases DT-EGF-induced cell death. Panel B, frames from a time-lapse microscopy experiment of U87MG cells treated with DT-EGF with or without 24 hours pre-treatment with trehalose to further increase autophagy; increased autophagy delays DT-EGF-induced death. Panel C, clonogenic survival assays in U87MG cells treated with DT-EGF in the presence or absence of trehalose to increase autophagy (mean +/- SD from three experiments). Increased autophagy inhibits DT-EGF-induced death. Panel D, caspase 3/7 activity in DT-EGF-treated U87MG cells after siRNA treatment (mean +/- SD from three replicates). Control and EGFR siRNA, DT-EGF-treated cells show no caspase activity, DT-EGF causes caspase activity in Atg5 siRNA-treated cells that is similar to the level achieved by treatment with Fas ligand plus cycloheximide.