performed. A final concentration of 100 mM etoposide (Sigma- Aldrich) was added approximately 24 hours after the transfection reagent was removed. After two hours, cells were washed with PBS and resuspended in DMEM plus 10% FBS and incubated at 37uC with 5% CO2 for 16 hours prior to cell death assays. Cell Death Assays. Cells were incubated with 9 nM Sytox Orange nucleic acid dye (Invitrogen) and 264 mM Hoechst 33258 at 37uC and 5% CO2 for 10 minutes prior to microscopy. Fluorescence and visible cell images were obtained using a Zeiss IM-35 epi-fluorescence microscope equipped with a CCD 300-T- RC camera (Dage MTI) controlled by Scion Image software. GFP and Sytox Orange fluorescence and phase images were superim- posed in Photoshop and live and dead cells were counted (see Figure S2 for representative images). Since N2A and H4 cell lines have low transfection efficiencies (,15%), the presence of EGFP or CT-EGFP was used to indicate transfected cells. A minimum of 50 cells was counted per sample and each condition was sampled at least three independent times. The mean value is plotted in the histograms with error bars representing the standard deviation. Statistics were performed using the Student’s T-test. Annexin V Staining. Approximately 24 hours after transfec- tion, cells were washed with annexin binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and stained with 5 mL Pacific blue-conjugated annexin V (Invitrogen) for 30 minutes at 37uC and 5% CO2. Cells were washed once with annexin V binding buffer, re-suspended in annexin V binding buffer with 9 nM Sytox orange, and incubated at 37uC and 5% CO2 for 15 minutes prior to assessment by fluorescence microscopy (see Figure S2 for representative images). The fraction of apoptotic cells were scored as GFP positive cells that were also Annexin V positive divided by the total number of GFP positive cells. Sytox orange was utilized to eliminate Annexin V positive cells that had a compromised membrane. A minimum of 50 cells was counted per sample and each condition was sampled at least three independent times. The mean value is plotted in the histograms with error bars representing the standard deviation. Statistics were performed using the Student’s T-test. Immunofluorescence. Approximately 24 hours prior to transfection, cells were plated onto glass coverslips. Cells were cotransfected with 1.5 mg each of pEGFPN-1 or CT-GFP and 352PHPtau/APPc31-myc using 3.5 mL Lipofectamine LTX and 1.0 mL Plus reagent according to the manufacturer’s instructions (Invitrogen). Approximately 4 hours after transfection, cells were washed with PBS, resuspended in DMEM with 10% FBS. Approximately 24 hours after transfection, the cells were washed in PBS and fixed for 10 minutes in 3.7% formaldehyde in PBS and 1.0 mM EGTA (pH 7.0). Cells were permeablized for 5 minutes in 0.1% Triton X-100 in PBS and 1 mM EGTA (pH 7.0). Cells were washed three times in PBS and blocked for 1 hour with 10.0% BSA in PBS. Cells were washed three times in PBS and incubated with mouse anti-myc 1:1000 (Cell Signaling) and rabbit References 1. Mott RT, Hulette CM (2005) Neuropathology of Alzheimer’s disease. Neuroimaging Clin N Am 215: 755–765. 2. Gibson PH, Tomlinson BE (1977) Numbers of Hirano bodies in the hippocampus of normal and demented people with Alzheimer’s disease. J Neurol Sci 33: 199–206. 3. Schochet SS, Jr., Lampert PW, Lindenberg R (1968) Fine structure of the Pick and Hirano bodies in a case of Pick’s disease. Acta Neuropathol (Berl) 11: 330– 337. 4. Yamamoto T, Hirano A (1985) Hirano bodies in the perikaryon of the Purkinje cell in a case of Alzheimer’s disease. Acta Neuropathol 67: 167–169. 5. Hirano A (1994) Hirano bodies and related neuronal inclusions. Neuropathol Appl Neurobiol 20: 3–11. 6. Mitake S, Ojika K, Hirano A (1997) Hirano bodies and Alzheimer’s disease. Kao Hsiung I Hsueh Ko Hsueh Tsa Chih 13: 10–18. Hirano Bodies Protect against Cell Death anti-FLAG 1:100 (Sigma) primary antibodies for 1 hour. After three washes in PBS, cells were incubated with Alexa 350-labeled goat anti-rabbit 1:200 (Invitrogen) and TRITC-labeled goat anti- mouse 1:700 (Sigma) secondary antibodies for 1 hour. Cells were washed three times in PBS and mounted with Crystal Mount (Biomeda). Images were obtained using a Deltavision microscope and subjected to deconvolution using Applied Precision software. Supporting Information Figure S1 Cell death and GFP expression levels are not affected by varying amounts of DNA during co-transfec- tion of multiple plasmids. H4 cells were transfected with pEGFPN-1 alone or with varying amounts of pcDNA3.1 to replicate co-transfection conditions with 1, 2, or 4 plasmids. Using flow cytometry, it was determined that the median fluorescence intensity of GFP in samples replicating co-transfection of 2 and 4 plasmids were not significantly different from GFP only transfected cells. Additionally, under these conditions cell death levels remained low, indicating that the cell death in our other experiments is not due to an increase in total DNA transfected. Error bar represents the coefficient of variation. (TIF) Figure S2 Representative images of the cell death quantitation. Live cells transfected with (A) GFP, (B) CT-GFP, GFP/APP/c31 stained with sytox orange (C) or (D) annexin V and Sytox Orange to mark apoptotic cells and rule out compromised membranes, respectively. Green = GFP, Blue = Hoescht 33258 (A and B), annexin V (D). Yellow = GFP and sytox orange. Arrows in C indicate nuclei stained with Sytox Orange. Scale bar = 20 mm. (TIF) Method S1 (DOCX) Acknowledgments Flow cytometry. The authors thank Drs. T. Su¨dhof (Stanford) and B. Hyman (Harvard) for generously providing the APP, APPc58-myc, Tip60 and Fe65 plasmids. The authors thank Dr. R. Brandt (University of Osnabru¨ck) for the generously providing the 352WTtau and 352PHPtau plasmids. The authors acknowledge the University of Georgia Coverdell Core Micros- copy Center for access to the Deltavision microscope. Author Contributions Conceived and designed the experiments: M. Furgerson M. Fechheimer RF. Performed the experiments: M. Furgerson. Analyzed the data: M. Furgerson RF M. Fechheimer. Contributed reagents/materials/analysis tools: M. Furgerson RF M. Fechheimer. Wrote the paper: M. Furgerson RF. 7. Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, et al. (1999) Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J Neurosci 19: 8876–8884. 8. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, et al. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long- term potentiation in vivo. Nature 416: 535–539. 9. Westerman MA, Cooper-Blacketer D, Mariash A, Kotilinek L, Kawarabayashi T, et al. (2002) The relationship between Abeta and memory in the Tg2576 mouse model of Alzheimer’s disease. J Neurosci 22: 1858–1867. 10. Cacucci F, Yi M, Wills TJ, Chapman P, O’Keefe J (2008) Place cell firing correlates with memory deficits and amyloid plaque burden in Tg2576 Alzheimer mouse model. Proc Natl Acad Sci U S A 105: 7863–7868. 11. Pimplikar SW (2009) Reassessing the amyloid cascade hypothesis of Alzheimer’s disease. Int J Biochem Cell Biol 41: 1261–1268. PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44996