Representative DRAQ5 and MAP2 images are shown in panels O and P. paraquat only elicited additive effects 24h after the final hit and even loss of warmth shock protein 70 activity HMOX1 and glutathione did not promote stress synergy at this early timepoint. Dual hits of MG132 elicited moderate glutathione loss and slightly synergistic toxic effects 48h after the second hit, but only at some concentrations and only relating to two viability assays (metabolic fitness and cytoskeletal integrity). The thiol N-acetyl cysteine safeguarded hippocampal neurons against dual MG132/MG132 hits but not dual MG132/paraquat hits. Our findings support the look at that proteotoxic and oxidative stress propel and propagate each other in hippocampal neurons, leading to synergistically toxic effects, but not as the default response and only after a delay. The neuronal stress synergy observed here lies in contrast to astrocytic reactions to dual hits, because astrocytes that survive severe proteotoxic stress resist additional cell loss following second hits. In conclusion, we present a new model of hippocampal vulnerability in which to test treatments, because neuroprotective treatments that are effective against severe, synergistic stress are more likely to succeed in the medical center. 5 (DIV5) for 24h. This was referred to as the 1st hit and was added to the existing press like a 10 answer. On DIV6, press were completely eliminated and cultures were Roxatidine acetate hydrochloride treated with new MG132 or paraquat inside a 1 answer. This DIV6 protocol facilitated the complete removal of the 1st hit and was referred to as the 2nd hit. Twenty-four or 48h later on, on DIV7 or DIV8, cell viability was measured as explained below. Wherever indicated, the heat shock protein 70 / warmth shock cognate 70 (Hsp70/Hsc70) inhibitor VER155008 (R&D Systems, Minneapolis, MN; Massey et al., 2010; Schlecht et al., 2013) or the glutathione synthesis inhibitor buthionine sulfoximine (Griffith, 1982) was Roxatidine acetate hydrochloride applied concurrently with MG132 and paraquat. Viability Assays Viability was measured using immunocytochemistry for the specific neuronal marker microtubule connected protein 2 (MAP2) with the infrared In-Cell Western technique, as published (Posimo et al., 2013; Posimo et al., 2014). Glutathione levels were measured in the same manner, according to published protocols (Posimo et al., Roxatidine acetate hydrochloride 2013; Titler et al., 2013; Unnithan et al., 2012). Roxatidine acetate hydrochloride Main antibodies are outlined in Supplemental Table S1. Infrared secondary antibodies were then applied to visualize MAP2 or glutathione (LI-COR Bioscience, Lincoln, NE; Jackson Immunoresearch Laboratories, Pub Harbor, ME). Immunolabeled cultures were also stained with the infrared nuclear stain DRAQ5 (1:10,000; Biostatus, Shepshed, Leicestershire, UK) for the second viability assay. All infrared staining was analyzed on an Odyssey Imager (Version 3.0, LI-COR Bioscience). Like a third viability measure, levels of ATP were measured with the CellTiter Glo assay (Promega, Madison, WI), as previously explained (Posimo et al., 2013; Posimo et al., 2014). In order to determine the neuronal purity of the cultures, cells were immunocytochemically labeled for the neuronal marker MAP2 and the astrocyte marker glial fibrillary acidic protein (GFAP) using visible-range secondary antibodies for higher resolution microscopy, as previously explained (Crum et al., 2015; Posimo et al., 2015). Roxatidine acetate hydrochloride For the second option experiments, nuclei were stained with Hoechst 33258 (10 g/mL, bisBenzimide) in phosphate-buffered saline with 0.3% Triton-X for 15 min. Photomicrographs were captured with an epifluorescent microscope (EVOS, Existence Systems) using the 20 objective (0.213 mm2 field of look at, three fields per well). An observer then counted the numbers of MAP2+ cells and Hoechst+ cells to determine neuron denseness in hippocampal cultures. Statistical Analyses Each experiment was run in at least three triplicate wells. The data from these three wells were averaged to yield an n of 1 1. Data are therefore.