Figure 6 See previous page.

Inhibition of autophagy reduces caspase activation and cell death in apoptosome-compromised cells exposed to sustained ER stress. casp9^−/− MEFs stably expressing pGIPZ or Atg5 shRNA were generated and treated for the indicated times with (A) 0.5 μM Tg or (B) 0.5 μg/ml of Tm after which lysates were assessed by immunoblotting for ATG5, cleaved CASP8, cleaved CASP3 and actin. (C) Atg5 shRNA and pGIPZ casp9^−/− MEFs were treated for the indicated times with Tg after which cells were fixed and stained with H&E. Representative images are shown. (D) casp9^−/− MEFs stably expressing pGIPZ or Atg5 shRNA were treated with 0.5 μg/ml of Tm for 72 h. Treatment was washed off and allowed to form colonies for 10 d. Colonies were stained with crystal violet and pictures taken. (E-F) casp9^−/− Atg5 shRNA and casp9^−/− pGIPZ MEFs were treated for the indicated times with (E) 0.5 μM Tg or (F) 0.5 μg/ml of Tm and cell viability analyzed by propidium iodide (PI) uptake. (G) pLKO and Atg7 shRNA casp9^−/− MEFs were treated for the indicated times with 0.5 μg/ml of Tm, cell lysates prepared and immunoblotted for cleaved CASP8, cleaved CASP3 and actin. (H) pLKO and Atg7 shRNA casp9^−/− MEFs were treated for the indicated times with 0.5 μg/ml of Tm and cell viability analyzed by propidium iodide (PtdIns) uptake. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD.