Exosomal secretion of AP/APP and BACE1 PCA reporter proteins.

<p>(A) Detection of AP/APP-BACE1 PCA signal in cleared, cell- and debris-free conditioned media. N2A cells were transiently transfected with BACE1-GLuc1 and AP/APP-GLuc2. Cells were incubated in serum-free media for up to 18 hours. PCA signals in cell-free conditioned media were detected at indicated time points. Normalization of cell numbers and transfection efficiency was done with an internal vector control. The values are normalized bioluminescence signals recorded from expressed pair of constructs. The number of replicate wells was four. The linearity was evaluated by regression analysis, correlation coefficient (R²) is 0.9971. Error bars represent the SEM. (B) N2A cells were transfected with AP/APP-GLuc2 and BACE1-GLuc1 reporter plasmids. Exosomes were isolated from 30-h conditioned media (inset graph shows data up to 60 h) by ultracentrifugation and the presence of AP/APP-GLuc2 and BACE1-GLuc1 reporters in exosomes was analyzed by Western blotting using antibodies for APP (C-terminal antibody A8717), dNGluc (BACE1) and Alix as an exosomal marker. Total cell extracts were analyzed in parallel with the isolated exosome fraction. (C) Pharmacological modulation of ceramide levels alters exosomal secretion of AP/APP-BACE1 PCA reporters. N2A cells were transiently transfected with BACE1-GLuc1 and AP/APP-GLuc2, and treated with 10 µM of GW-4869, the neutral sphingomyelinase (nSMase) inhibitor, for 24 h. PCA signal in cell-free conditioned media was measured at 48 h after transfection. Normalization of cell numbers and transfection efficiency was done with an internal vector control. The average values are displayed as percentage of change as compared to vehicle-treated control cells. Error bars represent the SEM, and statistical significance was assessed using Student's t test (four replicate wells/experiment, four independent experiments). *** p<0.001.</p

Multiplex assay for simultaneous live-cell detection of APP-BACE1 interaction and proteolytic cleavage of APP.

<p>(A) Graphical presentation of the multiplex PCA reporter constructs. Alkaline phosphatase (AP) with a signal peptide was placed in the N-terminus of APP-GLuc2. AP reporter is depicted in beige and GLuc reporter in red color. The same colors are used in column graphs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098619#pone-0098619-g003" target="_blank">Fig. 3B–F</a> for corresponding reporter data. (B) Normal proteolytic processing of the AP/APP-hGLuc fusion protein in N2A cells. Cells were transiently transfected with indicated combinations of various APP constructs: APP-GLuc2 and AP/APP-GLuc2 and BACE1-GLuc1. Western blots were probed with APP C-terminal antibody (A8717), dNGluc (BACE1) and GAPDH as a loading control. (C) Sensitivity and linearity of secreted alkaline phosphatase-sAPP (SEAP) assay from conditioned media. N2A cells were transiently transfected with BACE1-GLuc1 and AP/APP-GLuc2. Cells were incubated in serum-free media for up to 30 hours (inset graph shows data up to 60 h). AP activity in cell-free conditioned media was detected using a chemiluminescent SEAP assay. Normalization of cell numbers and transfection efficiency was done with an internal vector control (using a plasmid expressing β-galactosidase). The values are normalized chemiluminescence signals recorded from expressed pair of constructs. The number of replicate wells was four. Linearity of data was evaluated by regression analysis; correlation coefficient (R²) was 0.98806. Error bars represent the SEM. (D) Effects of brefeldin A (BFA) in the multiplex assay (PCA+AP data). N2A cells were transiently transfected with BACE1-GLuc1 and AP-APP-GLuc2, and treated with indicated concentration of BFA for 24 h before measurement of PCA and SEAP signals (48 h after transfection). The average values are displayed as percentage of change as compared to vehicle-treated control cells. (E) Effects of BACE inhibitor IV in the multiplex assay (PCA+AP data). N2A cells were transfected as in D, and treated with indicated concentration of BACE inhibitor IV for 6 h before measurement PCA and SEAP signals (48 h after transfection). The average values are displayed as percentage of change as compared to vehicle-treated control cells. (F) Effects of dynole 34-2 in the multiplex assay (PCA+AP data). N2A cells were transfected as in D, and treated with indicated concentration of dynole 34-2 for 6 h before measurement PCA and SEAP signals (48 h after transfection). The average values are displayed as percentage of change as compared to vehicle-treated control cells. Error bars represent the SEM, and statistical significance was assessed using Student's t test (four replicate wells/experiment, four independent experiments). * p<0.05, ** p<0.01, *** p<0.001.</p

Functional assay validation of GLuc PCA for APP-BACE1 interaction.

<p>For genetic assay validation of BACE1-APP PCA, plasmids expressing GGA3 and VPS35 shRNA were cotransfected to N2A cells with plasmids encoding BACE1-GLuc1 and APP-GLuc2 reporters. (A) PCA signal was measured at 48 h post-transfection. (B) Aβ₄₀ and Aβ₄₂ in conditioned media were determined by sandwich ELISA. The number of replicate wells for PCA was four (96-well plate) and for Aβ ELISA two (6-well plate). Error bars represent the SEM, and statistical significance was assessed using ANOVA. * p<0.05, ** p<0.01, *** p<0.001.</p

Protein-fragment complementation assay-based detection of APP and BACE1 interaction in live cells.

<p>(A) Graphical presentation of PCA reporter constructs of APP and BACE1. hGLuc, humanized <i>Gaussia</i> luciferase fragment; SP, signal peptide; TM, transmembrane domain. (B) Normal expression and proteolytic processing of APP-GLuc2 and BACE1-GLuc1 fusion proteins in N2A neuroblastoma cells. Cells were transiently transfected with indicated combinations of expression constructs and analyzed for APP fragments and BACE1 protein in cell lysates. Western blots were probed with APP C-terminal antibody (A8717), dNGluc antibody (detects the GLuc1 fragment) and GAPDH as a loading control. (C) Validation of GLuc-based PCA for detection of APP-BACE1 interaction in N2A cells. Cells were transiently transfected with indicated combinations of expression constructs. Luminescence signal was measured 48 h post-transfection in live cells. Normalization of cell numbers and transfection efficiency was done with an internal vector control (using a plasmid expressing β-galactosidase). Control plasmids were empty GLuc1/2 plasmids expressing the indicated GLuc fragment alone. The values are normalized bioluminescence signals recorded from expressed pairs of reporter constructs. Error bars represent the SEM, and statistical significance was assessed using ANOVA. *** p<0.001.</p