Figure 4

ARF4 knockdown protects from Golgi-stress-induced cell death and induction of DR4/5. (a,b) A549 cells were stably transduced with different shRNA constructs targeting either control genes (Ctrls; GFP or Luciferase) or ARF4 and tested for their sensitivity to either BFA (left panels) or thapsigargin (THA; right panels) by treating the cells for 48 h with increasing concentrations of these compounds. Afterwards, their relative viability was determined by a CTB assay (a), while LDH release in the culture supernatant was used as a relative measure of cell death (b). Relative values ((treated/untreated) for CTB, ((treated/untreated)−1) for 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 response of the experimental KDs was significantly different from the control KDs. *P<0.05, **P<0.01,***P<0.001. (c–e) To determine the induction of DR4 and DR5, control and ARF4 KD A549 cells were treated with vehicle (EtOH; 0) or increasing concentrations of either BFA or thapsigargin (THA). LDH release in the culture supernatant was determined after 48 h as a relative measure of cell death (c) and samples were prepared for western blot analysis to determine the expression levels of the indicated proteins (d,e). Blots were re-probed for β-Actin as a loading control. Black arrowheads indicate full-length proteins and their splice variants, white arrowheads their cleavage products or modified species of the protein. Blots are representative of three independent experiments