Figure 6

Induced expression of DR4 by Golgi stress sensitizes A549 cells to TRAIL. (a,b) A549 cells stably transduced with specific shRNA constructs targeting either a control gene (Luciferase), DR4, DR5 or both were exposed to increasing concentrations of TRAIL alone (a) or TRAIL plus 30 nM BFA (b) for 48 h. TRAIL was added 6 h after the addition of BFA or medium. Afterwards, relative cell viability was determined with a CTB assay (left) while LDH release in the cell culture supernatant was determined as a relative measure of cell death (right). 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 control KD upon treatment. *P<0.05, **P<0.01,***P<0.001. (c,d) A549 cells were incubated with increasing concentrations of BFA in the presence (black bars) or absence (white bars) of 25 ng/ml TRAIL for either 24 h (c) or 48 h (d). TRAIL was added 6 h after the addition of BFA or medium. Afterwards, LDH release in the cell culture supernatant was determined as a relative measure of cell death (left), relative cell viability was determined with a CTB assay (middle) while DEVDase activity was determined as a relative measure of caspase-3/7 activity, indicative of apoptotic cell death. Relative values (treated/untreated; CTB) or ((treated/untreated)−1; LDH release and DEVDase) represent the mean±S.D. of triplicate experiments