6-OHDA-triggered ROS induces upregulation of Puma. Dissociated dopaminergic cultures derived from C57Bl/6 mice were treated with 20 μM 6-OHDA and total RNA was collected at the indicated times. A) Levels of Puma and 18S rRNA were analyzed by RT-PCR. B) Gels in A were quantitated in ImageQuant and analyzed by one-way ANOVA (***, p < 0.001) with Bonferroni post-tests to compare each time point to untreated (9 hr and 12 hr: ***, p < 0.001). C) Cultures were treated with 5 mM NAC immediately prior to treatment with 20 μM 6-OHDA. Total RNA was collected and analyzed as in A. D) Gels in A were quantitated in ImageQuant and analyzed by two-way ANOVA (6-OHDA treatment: **, p < 0.01; NAC pre-treatment: ***, p < 0.001). E) Cell lysates were collected in RIPA buffer at the indicated times after treatment with 20 μM 6-OHDA. Protein levels were analyzed by western blotting for Puma. Antibody specificity was confirmed by comparing blots of known tissues positive and negative for Puma. F) Western blots in C were quantitated in ImageQuant. Levels of Puma were normalized to actin levels. Data were analyzed by one-way ANOVA (**, p < 0.01) with Bonferroni post-tests to compare each time point to untreated (24 hr: **, p < 0.01).