Puma mediates caspase-3 activation following 6-OHDA treatment. Dopaminergic cultures derived from Puma +/+ and Puma -/- animals were treated with 20 μM 6-OHDA. A) Cultures were fixed and stained at 0 or 24 hours for TH and cleaved caspase-3 (a-caspase-3). B) TH-positive and cleaved caspase-3-positive cells were counted and the percentage of TH-positive cells that were also cleaved caspase-3 positive was calculated. Data were analyzed by two-way ANOVA (treatment: **, p < 0.01; genotype: *, p < 0.05). C) Cell lysates were collected in RIPA buffer 24 hours after treatment with 20 μM 6-OHDA. Protein levels were analyzed by western blotting for cleaved caspase-3 and actin. D) Western blots in C were quantitated in ImageQuant. Levels of cleaved caspase-3 were normalized to actin levels. Data were analyzed by two-way ANOVA (treatment: **, p < 0.01; genotype: *, p < 0.05).