Figure 1

Tumor necrosis factor (TNF)-induced neurotoxicity and caspase activation in N27 dopaminergic neuronal cells. (A) Visualization of TNF toxicity by Sytox fluorescence cytotoxicity assay. N27 dopaminergic cells treated with TNF (30 ng/ml), or pretreated with etanercept (5 μg/ml) for 30 minutes and were processed for Sytox green imaging at 16 h. Increased green fluorescence is evident with TNF treatment, indicating significant neurotoxicity which was also evident in the phase contrast images. (B) Sytox fluorescence was quantified by measuring the fluorescence intensity as relative fluorescence units (RFU) using a plate reader. (C) Activation of caspase-8 by TNF treatment. Lysates from N27 cells treated with TNF (30 ng/ml) for 3 h were probed using a rabbit polyclonal antibody to caspase-8 that detects the procaspase and the active cleaved form. TNF induced a significant increase in the active caspase-8 (p20) fragment, which was blocked by pretreatment with etanercept (5 μg/ml). (D) Band intensities for cleaved caspase-8 from three blots were quantified using densitometric analysis and normalized to β-actin. (E) TNF induced activation of caspase-3. Enzymatic assays for caspase-3 activity in lysates from N27 cells treated with TNF (30 ng/ml) for 6 h or pretreated with either etanercept (5 μg/ml) or the caspase-8 inhibitor IETD-fmk (25 μM) for 30 minutes. Raw values were normalized using protein concentrations and expressed as percent control. TNF treatment induced significant activation of caspase-3, which was blocked by etanercept and the caspase-8 inhibitor IETD-fmk. (F) TNF-induced DNA fragmentation. N27 cells were treated with TNF (30 ng/ml) for 16 h or pretreated for 30 minutes with either etanercept (5 μg/ml) or the caspase-8 peptide inhibitor IETD-fmk (25 μM), and processed for the DNA fragmentation assay. Raw values were normalized using protein concentration and expressed as percent control. TNF treatment significantly increased DNA fragmentation, which was attenuated by etanercept or caspase-8 inhibition. Data represent the group mean ± SEM; n = 6 to 8 per group and experiments were repeated three times. ** (P <0.01) indicates significant differences compared to control cells; ## (P <0.01) and # (P <0.05) indicates significant differences compared to TNF-treated cells.