Figure 3

Knockdown of CHOP protects cells from secretory stress. (a–d) A549 cells (a,c) and HCT116 cells (b,d) stably transduced with shRNA constructs targeting control genes (Ctrls; Luciferase and GFP) or CHOP were tested for their sensitivity to BFA or thapsigargin (THA). Efficient KD of CHOP was verified by qPCR in the presence (overnight stimulation) or absence of THA (a,b), as indicated. Relative cell viability was assessed with a CTB assay 48 h after the addition of different concentrations of the indicated compounds (c for A549 cells and d for HCT116 cells). Relative values (treated/untreated) 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 expression of CHOP or the relative viability of the experimental KD was significantly different from the control KD. *P<0.05, **P<0.01,***P<0.001. (e–h): A549 cells (e,g) and HCT116 cells (f,h) stably transfected with different shRNAs targeting either CHOP or a control gene (Luciferase; Ctrl) were treated for 48 h with the indicated doses of BFA or vehicle (EtOH). Afterwards, lysates were prepared for western blot (e,f) and samples were taken of the culture supernatant to determine relative LDH release as a measure of cell death (g,h). Relative values ((treated/untreated)−1) represent the mean±S.D. of triplicate samples. Blots were probed for the indicated proteins and 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