Figure 11. PBA decreases ER stress induced apoptosis. The cells were treated with vehicle control (1:2000 DMSO) or the ER stress inducing agents: tunicamycin (5 mg/ml), thapsigargin (0.25 mM), or brefeldin A (5 mg/ml) in the presence or absence of PBA. A low and a high dose of PBA were used, 1 mM and 5 mM respectively, to assess concentration effects upon survival. The treatment was performed for 48 hours. After which, the detached cells were collected, spun down, and counted from triplicate plates on a standard hemocytometer. The number of detached cells increased by 3.8 fold (tunicamycin), 4.5 fold (thapsigargin), and 5.7 fold (BFA)—all of which were statistically different than basal levels (p,0.05) (A). With the addition of 1 mM PBA, the number of detached cells decreased to non-statistically significant levels in the tunicamycin and thapsigargin treated cells, while the numbers remained statistically different in the BFA treated cells. With 5 mM PBA co-treatment, the numbers of detached cells in all conditions were numerically and statistically indistinguishable from PBA Enhances APP Proteolysis basal levels (A). The pycnotic nuclei were scored by an independent scientist blinded to the conditions. A minimum of 1000 cells were counted in each condition. The fraction of pycnotic nuclei are represented relative to the total number of cells counted (% of total). All three ER stress inducing agents elicited a two-three fold increase in pycnotic nuclei. The fraction of cells with pycnotic nuclei decreased with PBA treatment—in the thapsigargin treated cells, the number of pycnotic nuclei was identical to the untreated cells by 1 mM PBA, and by 5 mM PBA the fraction of cells with pycnotic nuclei reached basal levels in all three treatment groups (B). doi:10.1371/journal.pone.0009135.g011 observed proteolysis. Increasing the concentrations beyond the levels demonstrated in Figure 12 did not enhance the stimulatory capacity of any of the compounds, and resulted in high levels of toxicity with both TUDCA and DMSO. This indicates that not all members of the small molecular chaperone class are equivalently effective in stimulating APP proteolysis, and that PBA may have some of its physiological effects through other biochemical mechanisms. Discussion The central question of this work is whether ER stress alters secretase mediated APP proteolysis. In order to address this question, we employed pharmacological treatment of the NAG cells, using agents known to alter protein processing within, or trafficking from, the ER. All pharmacological means of inducing ER stress result in the accumulation of misfolded proteins in the ER by distinct mechanisms. Our objective was to assess whether the general effects of ER stress alter APP processing; consequently, three different pharmacological stress inducing compounds are employed throughout this work—tunicamycin, thapsigargin, and BFA. Tunicamycin directly inhibits N-linked glycosylation of ER proteins, while thapsigargin targets SERCA-mediated calcium reuptake in the ER. Both of these approaches directly impact protein folding, as glycosylation-state promotes the transition from one conformational state to another—guiding the protein towards its native conformation [13,14]. Similarly, blocking calcium reuptake into the ER impairs chaperone function—as many of the ER chaperones use calcium as a co-factor, and consequently depend on intralumenal calcium to direct proper protein folding [13,14]. BFA directly targets the Arf-GTPase, and hence impairs ER to Golgi trafficking and promotes dissociation of the Golgi [33]. Our initial hypothesis purported that ER stress induced changes in trafficking would attenuate the levels of secretase mediated APP processing. In almost direct correspondence to UPR activation (Figure 2–4, blots) there was a partial arrest in APP proteolysis (Figures 2–4, bar graphs). As ER stress induction regulates both cellular transcriptional and translational responses, it could modify the reporter output associated with this assay system. However, there was no effect of ER stress induction upon bGal normalization or APPGV16 protein levels. Additionally, tran- siently transfected AICD-GV16, corresponding to the c-secretase mediated APPGV16 cleavage product, produced stable activation of the Gal4-reporter when treated with thapsigargin, tunicamycin or BFA (Figure S2). Consequently, the decrement in APPGV16 driven reporter activity with ER stress induction, represents a decrease in proteolytic liberation of the AICD-GV16 moiety. Consistent with other work [7,8], we find that induction of ER stress restricts APPGV16 localization to early components of the secretory pathway. As BFA blocks the trafficking of transmem- brane proteins out of the ER, the similarity in APPGV16 localization pattern in tunicamycin and thapsigargin treated cells PLoS ONE | www.plosone.org 13 February 2010 | Volume 5 | Issue 2 | e9135