(REMSA, Pierce Biotechnology, Rockford, IL). Briefly, rabbit ferritin light chain iron regulatory element (IRE) probe UCUUG- CUUCAACAGUGUUUGAACGGAAC was biotin labeled (Sig- ma, St. Louis, MO) at 39. 10 mg of cytoplasmic protein of organotypic slices following treatments was incubated with 2 nM (final concentration) of biotin labeled IRE probe in binding buffer provided in the kit following manufacturer’s protocol. 5 ml of IRP- IRE complexes formed were resolved on 5% DNA retardation gel (Biorad) and transferred on to nylon membrane (Roche Diagnos- tics, Indianapolis, IN). After the membrane was cross linked with UV-light, the IRP-IRE complexes were visualized with enhanced chemiluminiscence (Immun-star HRP chemiluminiscent kit, Biorad, Herculus, CA) according to the manufacturer’s protocol. Electrophoretic mobility shift experiments were performed at least three times and one representative experiment was shown. Reactive Oxygen Species (ROS) Assay ROS generation was determined using 29–79-dichlorofluores- cein-diacetate (DCFH-DA) and fluorometric detection of H2O2 as we have previously described [13]. For DCFH-DA measurements, 25 mg of homogenate from control and treated organotypic slices were diluted in PBS and incubated with 5.0 mM DCFH-DA (Sigma, St. Louis, MO) in the dark for 15 minutes at 37uC. Fluorescence was measured every 15 minutes for 1 hr with excitation and emission wavelengths of 488 nm and 525 nm respectively, using a Spectramax Gemini-EM (Molecular Probes, Sunnyvale, CA). Values are expressed as percent increase in fluorescence compared to controls. H2O2 was measured using the horseradish peroxidase (HRP)-linked fluorometric assay (Amplex Ultra Red; Invitrogen, Carlsbad, CA) following the manufacturer’s recommendation and as we described previously [13]. Resorufin fluorescence was followed by a Spectramax Gemini-EM (Molec- ular Probes, Sunnyvale, CA) with excitation 530–560 nm and emission at 590 nm. Oxidative stress measurements Isoprostanes and glutathione depletion assays were used to measure oxidative stress levels. The F2-isoprostane 8-Iso-prosta- glandin F2a (8-iso-PGF2a) is produced by lipid peroxidation and is a marker of oxidative stress. 8-iso-PGF2a levels were quantified in the control and treated organotypic slices using the isoprostane oxidative stress assay kit B (Biomol International, Plymouth Meeting, PA) as we have shown previously [13]. The color developed in the standards and samples was read on a SpectraMax Plus microplate reader (Molecular Devices, Sunnyvale, CA) at 405 nm. The measured optical density was used to calculate the concentration of 8-iso-PGF2a. Glutathione (gamma-glutamyl- cysteinyl-glycine; GSH) plays an important role in antioxidant defense in animal cells. Increased levels of oxidative stress lead to the accumulation of oxidized glutathione (GSSG) and a subse- quent decrease in the ratio of reduced glutathione (GSH) to GSSG. A luminescent based GSH-Glo Assay (Promega Corpora- tion, Madison, WI) was used for quantification of glutathione according to the manufacturer’s recommendation and as we have previously reported [13]. Confocal microscopy Alterations in total intracellular iron distribution were deter- mined using the fluorescent indicator Phen-green SK, the fluorescence of which is quenched by iron. Organotypic slices were incubated with phen green SK (20 mM of the diacetate) [14] for 1 hour at 37uC. The slices were mounted with vectasheild containing DAPI (Vector laboratories, Inc., Burlingame, CA). Phen-green SK staining was visualized with a Zeiss LSM 510 Gadd153 Mediates 27-OHC-Induced AD Pathology META confocal system coupled to a Zeiss Axiophot 200 inverted epifluorescence microscope. Imaging was performed with a 406 oil immersion objective. Statistical Analysis Data was analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test with GraphPad Prism software 4.01. All values in each group were expressed as mean value 6 SEM. All group comparisons were considered significant at p,0.05. Results Silencing gadd153 with siRNA reversed 27-OHC-induced increase in gadd153 levels Western blot and densitometric analysis (Fig. 1a,b) show that treatment with 27-OHC induced a substantial increase in gadd153 levels in comparison to control levels in hippocampal slices. Treatment with siRNA to gadd153 completely suppressed basal gadd153 levels and markedly reduced the 27-OHC-induced increase in gadd153 levels. Real-time RT-PCR demonstrated that 27-OHC also increases gadd153 mRNA and that treatment with siRNA to gadd153 reduces the basal as well as the 27-OHC- induced increase in gadd153 mRNA (Fig. 1c). Treatment of slices with siRNA to gadd153 reduced both the basal distribution of gadd153 and the large increase induced by 27-OHC treatment. Western blot and real-time RT-PCR demonstrate the efficiency of gadd153 silencing. Our results also show that the 27-OHC increased the levels of the ER-resident chaperone proteins grp 78 and grp 94 in organotypic slices (Fig. 1d–f). Treatment of slices with siRNA to gadd153 didn’t affect levels of grp 78 or grp 94 in slices untreated with 27-OHC but significantly reduced levels of these chaperones in slices treated with 27-OHC. Together, with increased gadd153, the increase in grp 78 and grp 94 levels demonstrates that 27- OHC targets the ER and causes stress in this organelle. siRNA to gadd153 reduced 27-OHC-induced increase in Ab levels Treatment with 27-OHC led to an increase in secreted levels of both Ab40 and Ab42 in media of the organotypic slices as determined with ELISA assay (Fig. 2a,b). While levels of Ab40 increased from 24.0560.79 in media of control slices to 31.3161.11 pg/ml in media of 27-OHC-treated slices, levels of Ab42 were 4.4960.59 pg/ml in media of control slices and 7.2660.55 pg/ml in media of 27-OHC treated organotypic samples. In media of slices treated with siRNA to gadd153 alone, levels of Ab40 and Ab42 were decreased by 45% and 52% respectively compared to control levels. siRNA to gadd153 also reduced levels of Ab40 and Ab42 by 36% and 54% in slices treated with 27-OHC compared to slices treated with 27-OHC alone (Fig. 2a,b). The decrease in Ab levels with siRNA to gadd153 suggests that gadd153 regulates the production of Ab from APP by BACE1 enzyme. To confirm the specificity of siRNA, we performed gadd153 silencing using the three siRNA probes individually, rather than a mixture of the three. Each of the three probes markedly reduced gadd153 as well as BACE1 levels (Supporting Figure S1). Our results show that the increase in the levels of Ab40 and Ab42 in the 27-OHC-treated slices was accompanied with an increase in the levels of parent protein APP as well as of BACE1, the enzyme that initiates the cleavage of APP to yield Ab (Fig. 2c– e). Treatment with siRNA to gadd153 alone, while didn’t affect APP levels, reduced BACE1 levels. Treatment with siRNA to PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26420