Materials and Methods Preparation of organotypic slices and treatments Organotypic slices were prepared as we have previously shown [8,10]. In brief, hippocampi from adult male rabbits (n = 4; 1.5–2 year old) were dissected and sectioned with a MacIlwain chopper (300 mm thick). Each hippocampus yields about 60 sections (120 sections per rabbit). Five sections were plated on membrane inserts (Millipore, Bedford, MD), with a total of 12 inserts per hippocampus (24 inserts per rabbit). Inserts were placed in 35 mm culture dishes containing 1.1 ml growth media (Neuroba- sal A with 20% horse serum, 0.5 mM l-glutamine, 100 U/ml penicillin, and 0.05 mM/ml streptomycin). Slices were exposed to a humidified incubator atmosphere (4.5% CO2 and 35uC). Media was changed at day 1, and at day 4 slices were switched to a defined medium consisting of Neurobasal A, 2% B27 supplement and 0.5 mM l-glutamine. At day 8, slices were divided to 4 groups (twenty four dishes of five slice each per group) as the following: Control slices, 27-OHC-treated slices, gadd153 siRNA-treated slices, and gadd153 siRNA+27-OHC-treated slices. All animal procedures were carried out in accordance with the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of North Dakota (protocol number 1101-1C). siRNA to gadd153/CHOP and non silencing control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The following human gadd153 gene sequences (59R39 orienta- tion) were used (A): Sense GAAGGCUUGGAGUAGACAAtt, Antisense UUGUCUACUCCAAGCCUUCtt; (B): Sense GGA- AAGGUCUCAGCUUGUAtt, Antisense UACAAGCUGAGAC- CUUUCCtt; (C): Sense GUCUCAGCUUGUAUAUAGAtt, Antisense UCUAUAUACAAGCUGAGACtt. The transfection of siRNA was performed in the slices with siRNA transfection reagent (Santa Cruz Biotechnology) and siRNA transfection medium (Santa Cruz Biotechnology) according to the manufac- turer’s recommendation on day 8. The siRNAs (final concentra- tion, 200 nM) were mixed with 100 ml of siRNA transfection medium. This mixture was gently added to a solution containing 20 ml of siRNA transfection reagent in 100 ml of siRNA transfection medium. The mixture solutions were incubated for 45 minutes at room temperature and 800 ml of siRNA transfection medium was added to the solution. Half of the final solution was added to the bottom of the inserts and remaining half onto the top of the slices with total of 1 ml in each dish. Transfected slices were incubated at 37uC for 48 hours without changing medium and then, treated with either 25 mM of 27-OHC (Medical Isotopes, Pelham, NH) or vehicle (0.1% ethanol). After 72 hours of treatment, samples were collected for ELISA, Western blot, real- time RT-PCR, reactive oxygen species, hydrogen peroxide, isoprostane, glutathione levels and confocal microscopy studies. Quantification of secreted Ab and TNF-a levels by ELISA Ab levels were quantified in the media of treated organotypic slices by ELISA using a kit from Invitrogen (Carlsbad, CA) as per the manufacturer’s protocol. Briefly, following treatments, the culture medium was collected, supplemented with protease and phosphatase inhibitor cocktail, and centrifuged at 16,0006 g for 5 min at 4uC. 100 ml of supernatant was used for Ab40 and Ab42 quantification. The quantity of Ab in each sample was measured in duplicate and expressed as mean 6 standard error for the samples. Ab40 and Ab42 levels are expressed in pg/ml. TNF-a released in media was quantified using a colorimetric sandwich human TNF-a ELISA (Invitrogen, Camarillo, CA) Gadd153 Mediates 27-OHC-Induced AD Pathology according to the manufacturer’s protocol. Treatments were performed in triplicate, and the quantity of TNF-a in each sample was measured in duplicate and expressed as mean 6 standard error for the samples. TNF-a levels are expressed in pg/ml. Western blot analysis Organotypic slices were homogenized with the tissue protein extraction reagent (T-PER, Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with the BCA protein assay reagent by standard protocol. Proteins (10 mg) were separated in 10% and 12.5% SDS-PAGE gels, transferred to a polyvinylidene difluoride membrane (Millipore) and incubated with antibodies to APP (1:100, Chemicon International, Temecula, CA), BACE1 (1:100, Chemicon International), phosphorylated tau (PHF-1 and CP13, 1:500, gift from Dr. Peter Davis, Albert Einstein College of Medicine), total tau (1:200, Calbiochem, La Jolla, CA), to the apoptosis-regulatory proteins Bax (1:100, Santa Cruz Biotechnol- ogy), caspase 3 (1:100, Santa Cruz Biotechnology) and Bcl-2 (1:100, Santa Cruz Biotechnology), to the ER- glucose regulated proteins grp 78 (1:100, Assay Designs Stressgen, Ann Arbor, MI) and grp 94 (1:100, Affinity Bioreagents, Golden, CO), to the ER- stress marker gadd153 (1:100, Abcam, Cambridge, MA), to the iron regulatory proteins transferrin receptor (TfR, 1:100, Abcam), ferritin light chain (FLC, 1:100, Santa Cruz Biotechnology), ferritin heavy chain (FHC, 1:100, Santa Cruz Biotechnology), and iron regulatory proteins-1 and 2 (IRP-1, 1:500, Millipore; IRP-2, 1:100, Chemicon International), and to the inflammation marker Tumor necrosis factor (TNF-a, 1:100, Abcam). b-actin (1:5000) was used as a gel loading control. The blots were developed with enhanced chemiluminiscence (Immun-star HRP chemiluminiscent kit, Biorad, Herculus, CA). The results were quantified by densitometry and represented as total integrated densitometric values. Real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) Total RNA was isolated and extracted from organotypic slices using the 5 prime PerfectPure RNA tissue kit (5 Prime, Inc., Gaithersburg, MD) as we have previously described [8]. 1 mg of purified RNA was converted to cDNA by using qScript CDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). The cDNA was amplified using PerfeCTa SYBR Green FastMix for iQ (2X, Quanta Biosciences) in Bio-Rad iCycler iQ Multicolor Real Time PCR Detection System (BioRad, Hercules, CA). Primers for gene of interest and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed with Beacon Designer 4.0. The primer sequences (forward; reverse, both in the 59 to 39 direction) are: Gadd153 (TGCTTCTCTGGCTTGGCTGAC; CTGGTTCTCCCTTGGTCTTCC), TNF-a (CGCTTCGCC- GTCTCCTACC; GGCAAGGTCCAGGTACTCAGG), and G- APDH (AGGTCATCCACGACCACTTC; GTGAGTTTCC- CGTTCAGCTC). The amplification process time was 15 min at 95uC followed by 80 cycles of 30 s at 95uC, 1 min at 55uC and 30 s at 72uC. Values were expressed in cycle threshold time (Ct) and were normalized to the Ct times for the housekeeping gene GAPDH. Primer specificity was confirmed by a single peak in a dissociation curve and/or by a single band following gel electrophoresis of the primer products. RNA band shift assay The IRP-IRE interactions were performed using LightShift Chemiluminescent RNA Electrophoretic mobility shift assay kit PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26420