Cathepsin D

For the methods used in this study, the following primary antibodies were utilized: MOAB-2 (anti-Aβ, mouse IgG_2b, 0.5 mg/ml), IgG_2b (0.2 mg/ml, Sigma-Aldrich, St. Louis, MO), 6E10 anti-Aβ residues 3-8, mouse IgG₁, 0.5 mg/ml; Covance, Princeton, NJ), 22C11 (anti-APP N-terminal, mouse IgG₁, 1 mg/ml, Milipore, Billerica, MA), 4G8 anti-Aβ residues 17-24, mouse IgG, Senetek, Maryland Height, MD), CT1565 (anti-APP C-terminal, rabbit monoclonal #1565, 0.45 mg/ml, Epitomics, Burlingame, CA), CT695 (anti-APP C-terminal, rabbit polyclonal #51-2700, 0.25 mg/ml, Invitrogen, Carlsbad, CA), anti-Aβ40 (MM32-13.1.1, mouse IgG₁, 1.8 mg/ml, Bu lab), anti-Aβ42 (rabbit, 0.35 mg/ml; Invitrogen, Carlsbad, CA), anti-β-actin (chicken, 1.0 mg/ml; Abcam, Cambridge MA) cathepsin-D (goat polyclonal, 0.2 mg/ml, Santa Cruz biotechnology, Santa Cruz, CA). The dilutions of each antibody stock are denoted in the appropriate Methods section or Figure Legend.

IDE activity was quantified by monitoring hydrolysis of FRET1 [31] (link), SP1 [23] (link), FAβB [17] (link) or ¹²⁵I-insulin [48] (link) as described previously, using recombinant human IDE purified from bacteria [33] (link). Degradation of unmodified recombinant human insulin (Sigma-Aldrich) and FITC-ins [28] (link) (Invitrogen) was assessed using a homogeneous time-resolved fluorescence (HTRF)-based assay (CIS-Bio). Quantitative kinetic data were derived by hyperbolic regression analysis using the computer program HYPER.EXE created by John S. Easterby (University of Liverpool). Activity of cathepsin B was assessed by monitoring hydrolysis of Z-LR-AMC (Enzo Life Sciences International), that of cathepsin D using the Cathepsin D Assay Kit (Sigma-Aldrich), and that of the 20S proteasome using the 20S Proteasome Assay Kit for Drug Discovery (Enzo Life Sciences International). The activity of all other proteases was assessed by monitoring the hydrolysis of OmniMMP™ Fluorogenic Substrate (Enzo Life Sciences International).

Mice were sacrificed and transcardially perfused with 4% paraformaldehyde (PFA) in cold phosphate-buffered saline (PBS). Mouse brains, superior cervical ganglions, and adrenal glands were collected, post-fixed in 4% PFA/PBS solution overnight, submerged in 30% sucrose in PBS for at least 72 h, and sectioned at 40 μm thickness using CM1950 cryostat (Leica)^{22 (link)}. Frozen sections were stained with antibodies specific to p150^Glued (amino acid 3–202 at the N-terminus of p150^Glued, BD Biosciences, #610474, 1:200, recognizing p150^Glued but not p135+), p150^Glued & p135+ (amino acid 1266–1278 at the C-terminus of p150^Glued, Abcam, #ab11806, 1:500, recognizing both p150^Glued and p135+), tyrosine hydroxylase (TH, Pel-Freez, #P40101-150, 1:2500; ImmunoStar, #22941, 1:500; Synaptic Systems, #213104, 1:500), dopamine transporter (DAT, Millipore, #MAB369, 1:500), vesicular monoamine transporter 2 (VMAT2, Synaptic Systems, #138302, 1:1000), glial fibrillary acidic protein (GFAP, Abcam, #ab7260, 1:1000), TAR DNA-binding protein 43 (TDP-43, Proteintech, #10782-2-AP, 1:500), α-synuclein (Santa Cruz, #sc-7011-R, 1:500; Santa Cruz, #sc-69977, 1:500), phosphorylated α-synuclein (Ser129) [p-α-synuclein (Ser129), Abcam, #ab51253, 1:500], neuronal nuclei (NeuN, Millipore, #ABN91, 1:500), synaptophysin (Millipore, #AB9272, 1:500), binding immunoglobulin protein (BiP, also referred to as GRP78, Abcam, #ab21685, 1:500), reticulon 3 (RTN3, Proteintech, #12055-2-AP, 1:500), 63 kDa cytoskeleton-linking membrane protein (CLIMP63, Proteintech, #16686-1-AP, 1:500), calnexin (Abcam, #ab22595, 1:500), protein disulfide isomerase (PDI, Proteintech, #11245-1-AP, 1:500), receptor binding cancer antigen expressed on SiSo cells (RCAS1, Cell Signaling Technology, #12290, 1:500), early endosome antigen 1 (EEA1, Cell Signaling Technology, #3288, 1:500), sequestosome 1 (SQSTM1, MBL, #PM066, 1:500), cathepsin D (R&D Systems, #AF1029, 1:500), ER-Golgi intermediate compartment 53 kDa protein (ERGIC53, Sigma-Aldrich, #E1031, 1:500), 130 kDa cis-Golgi matrix protein (GM130, BD Biosciences, #610822, 1:500), phosphorylated eukaryotic translation initiation factor 2α (Ser51) [p-eIF2α (Ser51), Abcam, #ab32157, 1:500], and phosphorylated inositol-requiring enzyme 1α (Ser724) [p-IRE1α (Ser724), Abcam, #ab48187, 1:500] as suggested by manufacturers. Alexa Fluor 488-, 546-, or 647-conjugated secondary antibody (Invitrogen, 1:500) was used to visualize the staining. Fluorescent images were captured using LSM 880 laser-scanning confocal microscope with Zen software (Zeiss) in conventional or Airyscan mode. As a high-resolution imaging modality, the Airyscan technology is reported to improve resolution 2-fold and signal-to-noise ratio 8-fold relative to the conventional confocal microscopy^{61 (link)}. The paired images in all the figures were collected at the same gain and offset settings. Post-collection processing was applied uniformly to all paired images. The images were presented as a single optic layer after acquisition in z-series stack scans at 1.0 μm intervals from individual fields or displayed as maximum-intensity projection or three-dimensional (3D) reconstruction to represent confocal stacks.

After CTL treatment for 24 h, breast cancer cells were fixed with 4% PFA in PBS and permeabilized with 0.5% Triton X-100 for 7 min. After PBS washing, cells were blocked with PBS-T containing 10% FBS and 1% BSA for 1 h. To measure the release of cathepsin D into the cytosol, cells were incubated with anti-cathepsin D primary antibody (sc-6486, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight and followed by Alexa Flour 488-conjugated anti-goat IgG for 1 h (1:250, Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 1 μg/mL DAPI (Sigma, St. Louis, MO, USA) in 2% BSA for 1 min. To analyze the mitophagy induction through the interaction of mitochondria with the lysosome, cells were incubated with primary antibodies (anti-COX Ⅳ, #4850, 1:250, Cell Signaling, Danvers, MA, USA and anti-LAMP2, sc-18822, 1:250, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were incubated with Alexa Flour 488-conjugated anti-rabbit IgG and 594-conjugated anti-mouse IgG (1:250, Invitrogen, Carlsbad, CA, USA) for 1 h. The autophagosome-mediated mitophagy was analyzed by staining with anti-COX Ⅳ in GFP-LC3-transfected cells, followed by incubating with Alexa Flour 594-conjugated anti-rabbit IgG. To assess the integrity of lysosome, cells were treated with CTL for 24 h and stained with 50 nM Lysotracker Red DND-99 at 37 °C for 30 min. Fluorescence images were acquired using confocal microscopy (Carl Zeiss, Jena, Germany).

Whole-cell lysates were prepared by adding 488 μl of radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl (pH 8.0), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 150 mM NaCl] to the wells after intracellular infection assays, where cells were scraped and kept in RIPA buffer for 30 min at 4°C. Before the addition of 74.5 μl of 1 M dithiothreitol and 187.5 μl of NuPAGE LDS sample buffer (4×) (Thermo Fisher Scientific), cells were further mechanically disrupted by passing the lysate through a 26-gauge size needle. Samples were then heated to 95°C for 5 min. Fifteen microliters of cell lysate proteins was then separated in a 4 to 12% (w/v) NuPAGE Bis-Tris protein gel and transferred to polyvinylidene difluoride membranes. Membranes were incubated with tris-buffered saline [50 mM tris and 150 mM NaCl (pH 7.5)] containing 0.1% (v/v) Tween 20 (TBST) and 5% (w/v) bovine serum albumin (BSA) for 1 hour at room temperature. Membranes were incubated with 1:1000 for rabbit α-cathepsin D (Cell Signaling Technology) or 1:1000 for rabbit α-GAPDH (Cell Signaling Technology) in TBST containing 1% (w/v) BSA overnight at 4°C. Membranes were washed for 60 min with TBST at room temperature and then incubated for 2 hours at room temperature with goat anti-rabbit (H+L) horseradish peroxidase–linked secondary antibody (Invitrogen). After incubation, membranes were washed with TBST for 30 min, and specific protein bands were detected by chemiluminescence using a SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific). Band intensities were quantified relatively to the lane’s loading control using ImageJ (52 (link)).