SK-N-SH cell lines were obtained from the ATCC (Rockville, MD, USA) and cell culture method has been previously described . The mouse neuronal cell lines ZW 13-2 and Zpl 3-4, which were established from the hippocampus of ICR (Prnp+/+) and Zürich I Prnp−/− mice, respectively, were kindly provided by Professor Yong-Sun Kim (Hallym University, Chuncheon, Kangwon-do, South Korea). The cells were grown in DMEM containing 10% FBS and gentamycin (0.1 mg/ml) in a humidified incubator maintained 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).
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 .
Immunocytochemical analyses were performed on neuroblastoma cells with p-nfkb (MAB3026; Milipore, Burlington, MA, USA) and anti-calcineurin (ab137335; Abcam). Cells were cultured on glass slides (Nalge Nunc International, Naperville, IL, USA), then washed in sterilized TBST for 10 min, blocked for 15 min with 5% FBS in TBST, and incubated overnight at 4 °C with the primary antibodies diluted with 5% FBS in TBST. Alexa Fluor 488-labeled donkey anti-rabbit IgG antibody, diluted 1:1000 (A21206; Molecular Probes, Eugene, OR, USA), was used to visualize expression using fluorescence microscopy.
Quantitative real-time PCR
Total RNA was extracted from SK cells using Easy-spin Total RNA Extraction Kit (iNtRON Biotechnology, Seoul, Korea). cDNA synthesis was carried out using TaKaRa Prime Script 1st Strand cDNA synthesis kit (TaKaRa Bio, Tokyo, Japan). For quantitative real-time PCR, 1 μl of gene-specific primers and SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) were used in 20 μl of reaction volume. The primers were as follows: TNF-α, forward = 5′TCT CCT TCC TGA TCG TGG C′ and reverse = 5′ GGT TCA GCC ACT GGA GCT 3′; COX-2, forward = 5′ AAA TTC GGT ACA TCC TCG AC 3′ and reverse = 5′ CAG GAA CTG GAT CAG GAC TT 3′; glyceraldehyde-3-phosphate dehydrogenase (as an internal control): forward = 5′ TGC CTC CTG CAC CAA CT3′ and reverse = 5′CGC CTG CTT CAC CTT C 3′. All quantitative PCR reactions were performed on a CFX96 real-time PCR detection system (Bio-Rad).
Western blot analysis
Western blot analysis was measured as described previously . The antibodies used for immunoblotting were anti-calcineurin (ab137335; Abcam), p-nf kb p65(Ser536) (#3033; Cell Signaling Technology, Danvers, MA, USA), p-bcl10, anti-P62 (#5114; Cell Signaling Technology), LC3 (Novus Biologicals, Littleton, CO, USA), and anti-β-actin (A5441; Sigma-Aldrich, St. Louis, MO, USA).
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.