Figure 7

Caspase-8 and CFLAR are required for Golgi stress-induced cell death. (a,b) A549 cells were either left untreated or treated for 48 h with increasing concentrations of BFA alone, 50 ng/ml TRAIL or BFA in combination with TRAIL. TRAIL was added 6 h after the addition of BFA. After treatment, the cells were lysed and analyzed on western blots probed for the indicated caspases, cleaved PARP as a relative indicator of cell death, the classical caspase-8 substrate Bid and re-probed for β-Actin as a loading control. Black triangles indicate full-length bands of the proteins or their splice variants, white triangles their cleaved fragments, where appropriate. In addition, images were collected with a light microscope with a × 10 objective (b). Inserts display a further × 2 magnification of the original pictures. For LDH release data, see also Supplementary Figure S7a. (c–f) A549 (c,d) or HCT116 cells (e,f) were stably transduced with different shRNA constructs targeting either control genes (GFP or Luciferase), CFLAR or caspase-8. Caspase-8 (c,e) or CFLAR (d,f) KD cells were treated with increasing concentrations of BFA for 48 h, after which relative viability was determined with a CTB assay (left panels) and relative LDH release was determined in the cell culture supernatant as a relative measure of cell death (right panels). Relative values (treated/untreated; CTB) or ((treated/untreated)−1; LDH release) represent the mean±S.D. of three independent experiments performed in triplicate for each cell line/condition. A two-way ANOVA was performed to determine whether the phenotype of the experimental KD cells was significantly different from the pooled control KDs after treatment. *P<0.05, **P<0.01,***P<0.001. (g,h) To determine caspase-8 activation upon stimulation with BFA as compared to TRAIL or TRAIL plus BFA, A549 and HCT116 cells were stimulated for 24 h with either 100 nM BFA, 100 ng/ml TRAIL (A549 cells), 10 ng/ml TRAIL (HCT116 cells) or BFA and TRAIL, as indicated. TRAIL was added 6 h after the addition of BFA. Afterwards, the cells were collected, lysed and the lysates subjected to immunoprecipitation of either caspase-8 (left) or CFLAR (right). Relative caspase-8 activity (LETDase) on the beads was determined with a caspase-8 GLO assay. Results represent the mean±S.D. of three independent experiments performed in triplicate