Translational suppression by V-siRNA precedes mRNA downregulation. Throughout this figure: red and orange, V-siRNAs to target; gray, V-blunt; green, vector-linked antisense to target; blue, unrelated V-siRNA; black, untreated control. a, SOD1 enzyme activity. Hippocampal cultures were treated with the indicated concentrations of vector-linked siRNA to SOD1 for 6 hr and assayed for SOD1 enzyme activity, given as mean ± SEM (n = 3). b, c, Western blots for SOD1 and caspase-3. Hippocampal cultures were treated with the indicated concentrations of V-siRNA for 6 hr and analyzed by Western blotting for targeted protein. Con, Control untreated; Bl, blunt-ended V-siRNA (see Results). Blots were stripped and reprobed for mouse inhibitor of apoptosis protein 2 (MIAP2) or actin to control for loading. SOD1 and caspase-3 levels are normalized to MIAP2 or actin and expressed as percentage of control, as determined by densitometry. d, V-SOD1i induces hippocampal neuronal death and does so more effectively than antisense oligonucleotides. Hippocampal cultures were treated with V-SOD1i-1/2, V-ASOD1, blunt-ended V-SOD1i-1 (V-SOD1bl), or unrelated vector-linked siRNA (V-M2i). Survival was assessed after 1 d by counting nuclei in cell lysates. This is a representative experiment; similar results were obtained in six independent experiments. Survival is reported relative to untreated cultures and is given as mean ± SEM (n = 3). e, V-Casp3i protects hippocampal neurons from HNE-induced death. Hippocampal cultures were treated with 10 μm HNE and simultaneously with V-Casp3i, antisense targeting caspase-3 (VACasp3), or blunt-ended V-Casp3i (V-Casp3bl). Survival was assessed after 1 d by counting nuclei in cell lysates (n = 3). This is a representative experiment; similar results were obtained in four independent experiments. Survival is reported relative to untreated cultures and is given as mean ± SEM (n = 3). f, g, Relative expression of SOD1 and caspase-3 mRNA after exposure to V-siRNAs. Hippocampal cultures were treated with the indicated constructs, and RNA was harvested after 6 (open bars) and 24 hr (filled bars). cDNA was analyzed by real-time reverse transcription PCR using serial dilutions. Each sample was analyzed in three independent runs. Results were normalized to α-tubulin mRNA levels and are given as the mean ± SEM (n = 3). Similar results were obtained when normalizing against caspase-2 mRNA levels.