SK-N-SH cell lines were obtained from the ATCC (Rockville, MD, USA) and cell culture method has been previously described . Cells were incubated at 37°C with 5% CO2.
PrP (106-126) treatment
Synthetic PrP (106-126) was synthesized as previously described . Sequences of PrP (106-126) used in this study were Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly. PrP (106-126) were synthesized by Peptron (Seoul, Korea).
Annexin V/Propidium iodide (PI) test
Apoptosis in detached 5,000 cells was assessed with an annexin V Assay kit to determine apoptosis according to the manufacturer's protocol. The staining procedure for PI and annexin V and incubated for 30 min in the dark. Flow cytometry was used to Guava EasyCyte HT System as described previously .
Measurement of [Ca2+]i
Measurement of Ca2+ contents was implemented as previously reported . Briefly, cells were plated on collagen-coated confocal dish. The SK-N-SH cells were loaded with 5 μM Fluo-4 AM (Invitrogen) for 40 min at 37°C. The image was processed using a confocal microscope (Zeiss). Fluorescence intensity was measured using Image J program (NIH).
Calcineurin activity assay
SK-N-SH cells were used to determine calcineurin phosphatase activity. Calcineurin activity was measured with calcineurin cellular activity assay kit (#BML-AK816-0001; Enzo Life Sciences, Inc., Farmingdale, NY, USA) and assay was performed following manufacturer's instructions. Calcineurin activity was measured as previously reported .
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.
Cellular apoptosis was measured using TUNEL assay (BioVision, Mountain View, CA, USA) and described previously .
Western blot analysis
Western blot analysis was measured as described previously . The 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).
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. Finally, these cells were visualized under a fluorescence microscope (#451203, Nikon ECLIPSE 80i; Nikon Corporation, Tokyo, Japan; magnification, x200 and fluorescence intensity was measured using Image J program (NIH).
Transmission electron microscopy (TEM)
TEM was performed as described previously . Briefly, SK-N-SH cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate, pH 7.2. Cells were post-fixed with 1% osmium tetroxide for further fixation. Specimens were dehydrated in graded ethanol and changed to propylene oxide, and then embedded in Epoxy resin. 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 statistical analysis was carried out using GraphPad Prism software (version 5.03; GraphPad Software, Inc., La Jolla, CA, USA). All experiments were expressed as mean ± standard error. And all experiments were compared using the one-way ANOVA followed by the Tukey test. Results were considered significant at * p < 0.05, ** p < 0.01 or *** p < 0.001, as appropriate.