Fig. 2.

Neuroprotective effect of human α-syn against oxidative stress without cellular toxicity. PC12/TetOn cells were seeded in 96-well plates at 100×10³ cells/well and grown overnight. Next day, cells were exposed to doxycycline 0.1 μg/ml or 1 μg/ml for 48 h. Afterward, H₂O₂ 150 μM was added for further 72 h and then cell viability was assessed by MTT or cells were fixed with 4% PFA and stained with 0.05% thioflavin-T as described in methods. (A) Viability of PC12/TetOn cells expressing different levels of human α-syn(WT) challenged by H₂O₂; (B) Viability assay of PC12/TetOn expressing α-syn(WT) after addition of doxycycline 1 μg/ml for increasing time intervals; (C) Evaluation of viability of PC12/TetOn expressing different levels of human α-syn(A30P) after oxidative challenge by H₂O₂. (D) Viability of PC12/TetOn stimulated with doxycycline 1 μg/ml to express human α-syn(A30P) for increasing time intervals. *P<0.05; **P<0.01, one-way ANOVA followed by Tukey's post-hoc test. (E) Absence of α-syn aggregate formation assessed by thioflavin-T staining in PC12/TetOn cells after 48 h stimulation with doxycycline 1 μg/ml. The upper pictures show the basal condition (−doxy), while the +doxy conditions are the stimulated cells. (F) Representative dot blot assessing A11 reactivity in uninduced (−doxy) and induced (+doxy) condition for both cell lines at two different time-points (48 and 96 h) from doxycycline addition. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.