antibodies were previously discussed. APPGV16 was visualized using the rabbit VP16 antibody and the Donkey anti-rabbit Alexa 546 (Molecular Probes) secondary. DAPI (Vectashield) or Hoescht nuclear stains were used to resolve the nuclei of the cells. The pycnotic nuclei were identified in the apoptosis assays by counting the number of cells with either condensed or fragmented nuclei relative to the total number of cells in each condition by an observer blinded to the identity of the experimental conditions. No less than 1000 cells were counted per condition. Results Stably Transfected Neuroblastoma Cells Can Be Used to Measure c-Secretase Activity The neuroblastoma N2a APPGV16/Gal4-luciferaseEGFP/ EF1-NLS-bGal (NAG) cells were generated as described in the Materials and Methods section. The NAG cells employ a Gal4- driven luciferase-EGFP fusion reporter which quantitatively measures c-secretase cleavage of APPGV16. The APPGV16/ Gal4-reporter system was employed successfully as independent vector components in transiently transfected cells[5,29]; yet this is the first report of the reconstruction of this assay system into stably transfected neuroblastoma N2a cells. As a novel model, we validated that the Gal4-reporter output corresponds to c-secretase activity. We tested both the luciferase and the EGFP components of the Gal4-reporter system with the addition of the c-secretase inhibitor DAPT. The NAG cells treated with DAPT for 24 hours demonstrated almost no EGFP signal, which was robust in the untreated cells (Figure 1A). This validated the c-secretase dependence of the Gal4-EGFP output. Quantitative measures of the luciferase output in NAG cells treated with titrated concentrations of DAPT for 24 hours were employed to determine the c-secretase dependence of the raw luminometric output. These values were normalized to bGal activity. Consistent with the EGFP results, the luciferase activity decreased monotonically with increasing DAPT concentrations, with a maximal inhibition of 80% of Gal4-reporter activity (Figure 1B). The IC50 was approximately 1 mM DAPT, consistent with previous reports of DAPT inhibition of c-secretase [30,31]. DAPT did not impact the normalization values in the bGal assays, ruling out toxic or non- specific transcriptional effects. As occurs with endogenous APP, DAPT treatment resulted in elevated levels of the a- and b- secretase derived APP carboxy-terminal fragments (CTFa and CTFb), with CTFa being the predominant proteolytic species observed (Figure 1C). The holoAPPGV16 levels did not change across the DAPT titration, ruling out APPGV16 down-regulation, or variations in protein loading, contributing to the DAPT mediated decrease in reporter activity. Consequently, the Gal4- reporter system, stably incorporated into the NAG cells, is a valid measure of c-secretase mediated proteolytic processing of APPGV16. Endoplasmic Reticulum (ER) Stress Induction Results in Impaired APP c-Proteolysis and Induction of Apoptotic Signaling (Figures 2–4) As already mentioned, ER stress is implicated in AD pathogenesis; however, how it impacts c-secretase processing of APP is relatively unexplored. ER stress effects upon APP proteolysis were measured in NAG cells treated with titrated concentrations of three different compounds: tunicamycin, thapsigargin and brefeldin A (BFA). Tunicamycin is a well known inhibitor of N-glycosylation, preventing N-glycan maturation upon transmembrane proteins within the secretory pathway [13]. Thapsigargin is a sarcoplasmic/endoplasmic reticulum calcium PBA Enhances APP Proteolysis Figure 2. Thapsigargin inhibits APP proteolysis and induces ER stress UPR signaling. Increasing doses of thapsigargin inhibits c- secretase cleavage of APPGV16 (top graph). Titrated doses of thapsigargin induced graded increases in UPR stress signaling through phospho-PERK (pPERK blot) and phospho-eIF2a (p-eIF2 a), beginning at 0.1 mM and reaching a plateau at 0.5 mM thapsigargin. The increases in UPR signaling correlate with the levels of phospho-p38 (pp38 blot) and phospho-JNK (pp54/JNK, pp46JNK blot), as maximal stimulation is observed in the same concentration range. Total protein expression levels remain unchanged, as eIF2a and APPGV16 protein levels were consistent across treatment conditions. doi:10.1371/journal.pone.0009135.g002 ATPase (SERCA) pump inhibitor, blocking Ca2+-reuptake into the ER, and impairing calcium dependent chaperone function [32]. BFA blocks trafficking through the Golgi by targeting the PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9135