Zou and colleagues demonstrated that HSF1 can be cross-linked to Hsp90 in unstressed HeLa cells, suggesting that HSF1 might interact with Hsp90

se, 25; KH2PO4, 10; Tris-HCl, 10; and ADP, 0.1, with or without 0.51 mM glutamate. In preliminary experiments these glutamate concentrations gave the maximal response in terms of stimulation of ATP synthesis without toxic effects in our systems. The tested drugs or the respective vehicles were added to mitochondrial suspensions 15 min before glutamate stimulation and throughout the experiments. Luminescence was measured with a luminescence counter. All experiments were performed using,60 mg of mitochondria, an amount that in preliminary tests ensured strong and reproducible ATP signal, and 1 h incubation, which in the same preliminary tests provided the best Mitochondrial NCX1/EAAC1 Sustain Brain Metabolism compromise between mitochondrial viability and ATP accumulation. The magnitude of ATP response to glutamate alone was found to vary between mitochondrial preparations. Therefore, for each set of experiments, for each experimental session, every batch of mitochondrial preparations was used and divided in the following groups: unstimulated, stimulated with glutamate, treated with tested drugs 6 glutamate, as indicated. Cultured cells. Cells, 24 h after been plated in 96 multiwell plates, were transfected with ODNs for additional 24 h for NCX1 or EAAC1 and 48 h for Citrin/AGC2 knock-down experiments. Afterward they were first washed with standard buffer solution containing: NaCl, 140; KCl, 5; CaCl2, 1; MgCl2, 0.5; HEPES, 10; and glucose, 5.5, pH 7.4, adjusted with Tris, and then exposed to glutamate in standard buffer solution for 1 h at 37uC. ATP levels were analyzed after incubation. All ATP data were normalized to the protein content of the different preparations. Statistical analysis Data were expressed as mean 6 SEM. p,0.05 was considered significant. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. For experiments performed in pair-wise fashion, significance of results was determined by Student’s t-test. Drugs and chemicals DL-TBOA, -3-amino]phenyl]methoxy]-L-aspartic acid and CGP-37157 were obtained from Tocris. Ru360 was dissolved in ultra pure distilled water at final concentration of 8663121 10 mM and used immediately after. All the other chemicals were of analytical grade and were purchased from Sigma. Supporting Information ODN experiments Chimeric phosphorothioated sense and antisense oligonucleotides against rat or human EAAC1 and a mixture of two chimeric phosphorothioated sense and antisense ODNs against rat or human Citrin/ AGC2 were designed to target the area near the start region of the genes and used as reported previously to knock down respectively EAAC1 and Citrin/AGC2. SH-SY5Y or C6 cells were transfected with Attractene or Lipofectamine 2000 using standard TSU68 chemical information protocols. Transfection efficiency was quantified using a plasmid encoding the enhanced green fluorescent protein cotransfected with ODNs. The efficiency of the gene silencing obtained with AsODNs was measured by real-time PCR and averaged at 60%, both for EAAC1 and for Citrin/AGC2. Chimeric phosphorothioated sense and antisense ODNs were used to knock down the different NCX subtypes as described previously. The protein levels of NCX1, EAAC1 and ACG2 were selectively decreased in SH-SY5Y and ” C6 cells treated with the respective AsODN. As-ODNs used to specifically knock-down only one of the three transporters were without effect on the protein levels of the two other ones. PCR analysis Real-time PCR was performed as described p