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(a) Effect of NAC on MA-induced MDC accumulation
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(b) Effect of NAC on MA-induced upregulation of LC3-II
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(c) Effect of BSO on MA-induced upregulation of LC3-II
Figure 3: (a) Effect of NAC on MA-induced accumulation of monodansylcadaverine (MDC) in autophagy vacuoles. N27 dopaminergic cells were preincubated with 5 mM NAC for an hour prior to exposure to MA (2 mM) for 18 hours. MA-induced changes in intracellular MDC fluorescence were measured as indicated in Methods. The data represent mean ± SEM of four individual measurements. Asterisks (* and ** ) indicate significant differences between MA-treated cells and NAC alone or NAC with MA-treated cells. Values are expressed as percentage of MDC specific activity compared to untreated control cells. Treatment of N27 dopaminergic cells with 20 μM chloroquine is considered as test control. (b) NAC reduced LC3-II levels in N27 dopaminergic cells treated with MA. Western blot analysis of LC3-II expression in N27 dopaminergic cells after exposure to MA (2 mM) with or without 5 mM NAC during 18 hour treatment is presented. Equal loading of protein in each lane is confirmed by probing the membrane with β-actin antibody. Densitometry analysis of LC3-II induction ( ) is represented next to the Western blot image. (c) BSO enhances the expression of LC3-II. N27 dopaminergic cells were pretreated with 100 μM BSO for 24 hours and treated with MA for another 18 hours. Equal loading of protein in each lane is confirmed by probing the membrane with β-actin antibody. Densitometry analysis of LC3-II induction is represented next to the Western blot image. LC3-II bands were quantified and expressed as percentage of untreated control. Data represent ± SEM, . * , ** , and *** compared with MA-treated cells.