The human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Minimum Essential Medium (MEM, Hyclone Laboratories, Logan, UT, USA) containing 10% fetal bovine serum (Invitrogen-GIBCO, Grand Island, NY, USA) and gentamycin (0.1 mg/mL) in a humidified incubator maintained at 37°C and 5% CO2.
PrP (106-126) treatment
Synthetic PrP (106-126) peptides (sequence, Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly) were synthesized by Peptron (Seoul, Korea). The peptides were dissolved in sterile dimethyl sulfoxide at a stock concentration of 10 mM and stored at -20°C.
Annexin V/Propidium iodide (PI) test
Apoptosis in detached cells was assessed using an annexin V Assay kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's protocol. Annexin V levels were determined by measuring fluorescence at 488 nm excitation and 525/30 emission using a Guava EasyCyte HT System (Millipore, Bedford, MA, USA).
Measurement of [Ca2+]i
Neuronal cells on collagen-coated confocal dish were incubated with 5 μ M Fluo-4 AM (Invitrogen) in media containing 1% FBS at 37°C for 40 min. The cells were washed three times with Hank’s Balanced Salt Solution (HBSS). Changes of [Ca2+]i were determined at 488 nm excitation/530 nm emission using an air-cooled argon laser system. The fluorescence emitted at 530 nm was collected using a photomultiplier. The image was scanned using a confocal microscope (Zeiss). For the calculation of [Ca2+]i, the method of Tsien et al.  was used based on the following equation: [Ca2+]i = Kd(F − Fmin)/(Fmax − F), where Kd is 345 nM for fluo-4, and F is the observed fluorescence level. Each tracing was calibrated for maximal fluorescence (Fmax) via addition of ionomycin (2 μM) and for minimal fluorescence intensity (Fmin) via addition of EGTA 5 mM at the end of each measurement.
Calcineurin activity assay
The calcineurin cellular activity assay kit (#BML-AK816-0001; Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used to determine the phosphatase activity of calcineurin in neuronal cells, using the manufacturer's instructions. In brief, the cells were lysed on ice in a lysis buffer containing protease inhibitors. Phosphatase activity was quantified by detection of free phosphate released from the reaction based on the absorbance of malachite green (OD 620 nm).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.
TUNEL assay was carried out to measure the stage of cellular apoptosis using a TUNEL-based assay kit (BioVision, Mountain View, CA, USA). TUNEL analysis was performed according to the manufacturer's instructions. PI was used to show the cell nuclei.
Western blot analysis
SK-N-SH cells were lysed in a lysis buffer [25 mM HEPES at pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 0.1 mM DTT (dithiothreitol), and a protease inhibitor mixture]. Whole cell proteins were electrophoretically resolved on a 10%–15% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected via sequential incubation with primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents (West Save Gold detection kit; AbFrontier, Seoul, Rep. of Korea). The primary antibodies used for immunoblotting were anti-P62 (#5114; Cell Signaling Technology), LC3 (Novus Biologicals, Littleton, CO, USA), and anti-β-actin (A5441; Sigma-Aldrich, St. Louis, MO, USA). Images were analyzed using a Fusion FX7 imaging system (Vilber Lourmat, Torcy Z.I. Sud, France). Densitometry was used to analyze the signal bands with the Bio-1D software package (Vilber Lourmat, Marne La Vallee, France).
After treatment, coverslips were mounted with 70% ethanol in DW for 10 min at RT. Cells were stained with AO (0.1 µg/mL) for 20 min in a cell culture incubator. Excitation wavelengths were 543 nm and 633 nm. Bandpass filters were set at 560–615 nm (Cy3, AlexaFluor568) and 650–750 nm (AlexaFluor647).
Transmission electron microscopy (TEM)
Following fixation of the cells in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2% paraformaldehyde (Electron Microscopy Sciences) in 0.05 M sodium cacodylate (pH 7.2; Electron Microscopy Sciences) for 2 h at 4˚C, specimens were fixed in 1% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4°C, dehydrated with increasing ethanol (25, 50, 70, 90 and 100%) for 5 min each and embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60˚C according to the manufacturers' instructions. Ultrathin sections (60 nm) were prepared using an LKB‑III ultratome (Leica Microsystems GmbH, Wetzlar, Germany) and were stained with 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were recorded on a Hitachi H7650 electron microscope (Hitachi, Ltd., Tokyo, Japan; magnification, x10,000) installed at the Center for University‑Wide Research Facilities (CURF) at Jeonbuk National University.
All data were expressed as mean ± standard error, and compared using the one-way ANOVA followed by the Tukey test. All statistical analysis was performed using GraphPad Prism software. Results were considered significant at * p < 0.05, ** p < 0.01 or *** p < 0.001, as appropriate.