MG132 induces caspase cascade activation in Cas-expressing cells. (A and B) Immunoblots showing cleavage of PARP (A) or caspases (B) in Cas-/- (EV), Cas-FL and WT MEFs treated with 1 or 5 μM MG132 for 16 hours as indicated. In the bottom panel in (A), Cas-FL cells were treated with 1 μM MG132 for times indicated, to assess time-course for PARP cleavage. (C) Caspase-3/7 activity assay was carried out using the Caspase-Glo® 3/7 Assay kit in MEFs treated with or without 1 μM MG132 for 16 hours. Sample luminescence was measured, and luminescence activity (RLU) was normalized by undertaking parallel MTS assays and evaluating cell number in untreated samples to reflect caspase activity. (D) Cas-/- (EV), Cas-FL and WT MEFs treated with 1 μM MG132 were lysed, and separated into cytosolic and mitochondrial fractions, followed by immunoblot to detect the release of cytochrome c from mitochondria to cytosol. Tubulin and VDAC served as cytosolic and mitochondrial markers, respectively to assess purity of the fractions. (E) Caspase-8 activity was determined using the Caspase-Glo® 8 Assay kit in MG132-treated Cas-/-(EV) and Cas-FL MEFs, and luminescence was examined as in C. (F) The caspase-8 inhibitor Z-IETD-FMK (25 μM) was used with various concentrations of MG132 to determine caspase-8 involvement in Cas-FL MEFs death, as measured by the MTS assay. (G) HeLa cells stably transfected with Caspase-8 shRNA or control shRNA were treated with the indicated concentrations of the proteasome inhibitors MG132 or Bortezomib for 16 hours, and cell viability was determined by MTS assay. shRNA-mediated reduction of Caspase-8 expression is shown in the immunoblot.