Expression of ATXN7Q65-GFP leads to increased ROS levels prior to toxicity. A) Western blot analysis of ATXN7 in FLQ10 and FLQ65 stable PC12 cell lines induced to express GFP-tagged ATXN7 with 10 (ATXN7Q10-GFP, top panel) or 65 (ATXN7Q65-GFP, lower panel) glutamines for the indicated number of days. Actin was used as loading control. B) Analysis of ATXN7 aggregation in FLQ65 cells induced to express ATXN7Q65-GFP for 1–12 days using the filter trap assay. C) Viability measured by the WST-1 assay and normalized against the protein content in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. D) Toxicity measured by the LDH membrane leakage assay in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. E) Total cellular ROS levels measured using DCHF-DA in cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for 0–12 days. F) Analysis of GSH levels in cells induced (−Dox) to express ATXN7Q65-GFP for 9 days and non-induced control cells (+Dox). For quantifications all data are shown as means ± SEM from three independent experiments with triplicates. * p <0.05, ** p <0.01 and *** p <0.001.
Ajayi et al. BMC Neuroscience 2012 13:86 doi:10.1186/1471-2202-13-86