Figure 5

Secretory stress leads to intracellular accumulation of DR4. (a,b) A549 cells were treated for 24 h with increasing concentrations of BFA (a) or thapsigargin (THA) (b). Afterwards, the cells were collected, fixed and divided over different samples. Half the samples were permeabilized (top) before probing with specific antibodies against either DR4 (left) or DR5 (right) to determine total (intracellular and membrane) expression of the DRs, the other half was left unpermeablized to determine the fraction of DRs exposed on the cell membrane (bottom). Data were collected by FACS analysis and representative histograms of three independent experiments are shown, depicting cell count over mean fluorescence intensity (MFI) expressed in arbitrary units (A.U.) as compared to staining with the isotype control antibody (Iso.). (c,d) A549 cells were grown on microscopy cover slips and incubated with either vehicle (EtOH), 100 nM BFA or 100 nM thapsigargin (THA) for 24 h. Afterwards, the cells were washed, fixed, permeabilized and probed overnight with specific antibodies against either the ER marker Calnexin (CNX; c) or the Golgi marker GM130 (d) in combination with an antibody against DR4. The following day, cells were washed again and probed with fluorescently labeled secondary antibodies to detect the localization of DR4 as well as Hoechst to stain the nuclei. Coverslips were then mounted on slides and analyzed by fluorescence microscopy. Representative images of single channels and overlays are shown