Cell culture
The human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Minimum Essential Medium (MEM, Hyclone Laboratories, Logan, UT, USA) with 10% fetal bovine serum (Invitrogen-GIBCO, Grand Island, NY, USA) and 0.1 mg/mL gentamycin in a humidified environment at 37°C with 5% CO2.
PrP (106-126) treatment
PrP (106-126) was synthesized as previously described [45]. 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 5,000 cells was assessed using an annexin V Assay kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's protocol. Cells were incubated in the dark for 30 min. 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
Measurement of Ca2+ contents was measured as previously reported [46]. Briefly, cells were plated on collagen-coated confocal dish. Incubation with 5 μ M Fluo-4 AM (Invitrogen) in media containing 1% FBS was carried out for 40 min at 37°C and then 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. [47] was used.
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. Calcineurin activity was measured as previously reported [48]. 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 performed to measure the degree of cellular apoptosis using a TUNEL-based assay kit (BioVision, Mountain View, CA, USA). TUNEL analysis was performed according to the manufacturer's instructions. The cells were counterstained with propidium iodide (PI) for 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]. Equal amounts of lysate 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).
Fluorescence microscopy
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)
TEM samples were analyzed by transmission electron microscope (JEM-2010, JEOL) installed in the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University [49]. Briefly, TEM samples were analyzed by transmission electron microscope (JEM-2010, JEOL) installed in the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University. After fixation of SK-N-SH cell samples in 2.5% glutaraldehyde (TED PELLA, USA) in PBS (pH,7.2), specimens were post fixed in 1% osmium tetroxide (Heraeus, South Africa), dehydrated in graded ethanol and propylene oxide (Acros Organics, USA), and then embedded in Epoxy resin (Embed812. NMA; Nadic methyl anhydride. DDSA; Dodenyl Succinic Anhidride. DMP-30., USA) as used previously. Serial ultrathin sections were cut on an LKB-III ultratome (LEICA, Germany). Ultrathin sections were stained with uranyl acetate (TED PELLA, USA) and lead citrate (TED PELLA, USA) and examined with the aid of a Hitachi H7600 electron microscope (Hitachi, Japan) at an accelerating voltage of 100 kV.
Statistical analysis
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.