Effects of SAHA on viability of human primary astrocytes and their IFN-γ-induced toxicity toward SH-SY5Y cells. Human astrocytes were incubated with or without SAHA at the concentrations indicated for 1 h before stimulation with IFN-γ (50 U/ml) for 48 h (A-F), or SAHA was added directly to cell-free supernatants from IFN-γ-stimulated astrocytes (B, C, right bars). Astrocytes in the control group were incubated with medium only. After 48 h incubation, cell-free supernatants were collected from astrocyte cultures and the astrocyte viability was measured by the MTT assay (A). The collected supernatants were transferred to cultures of SH-SY5Y cells and their viability assessed by the MTT (B) and LDH (C) assays 72 h later. To establish that SAHA does not neutralize neurotoxins in stimulated supernatants, 1 μM of SAHA was added into the supernatants from astrocytes stimulated with IFN-γ (50 U/ml) for 48 h just before applying such supernatants to SH-SY5Y cell cultures. After 72 h incubation, the SH-SY5Y cell viability was assessed by the MTT assay (B, right bar) or the LDH assay (C, right bar). A phase-contrast microscopy with 20x and 40x (see inserts) objectives was used to analyze morphology of SH-SY5Y cells (D-F). D, control; E, IFN-γ alone; F, 1 μM SAHA + IFN-γ. Every scale bar indicates 100 μm (D-F). Data (mean ± S.E.M.) are expressed as percent of live cells, where the 100% value is obtained from either non-stimulated astrocytes in the control group (A) or SH-SY5Y cells incubated with fresh medium only (B), or percent of lysed cells, where the 100% value is obtained from cells lysed by 1% Triton X-100 (C). *Significantly different from IFN-γ stimulation only. n = 6.