Neuroprotective effects of HO-1. A: HO-1 siRNA was used to knockdown HO-1 expression in mixed neuron and glial cultures. Control siRNA was used to control for nonspecific effects. Briefly, cultures were incubated with siRNA for 2 to 5 days before treatment with a cytokine mixture for an additional 3 days, as described in Materials and Methods. Vital dye exclusion test was used to determine the number of dead neurons as described in the Figure 3 legend. The results show that the neuroprotective effect of 3-HAA is diminished in the presence of HO-1 siRNA. **P < 0.01. NS = nonsignificant. Results are representative of three experiments using different donor cells. B: The effect of HO-1 siRNA on HO-1 protein expression was determined by HO-1 immunocytochemistry. In control siRNA-treated cultures (upper panel), many astrocytes show strong HO-1 immunoreactivity. In HO-1 siRNA-treated cultures, HO-1 immunoreactive cells virtually disappear (lower panel). C: The effect of CoPP, an inducer of HO-1, was examined in the neurotoxicity assay, and the results show that CoPP (1 μmol/L) protected neurons from cytokine-induced death. ***P < 0.001.