Effects of autophagy and caspase inhibitors on survival of neurons after glucose reperfusion (GR).

<p>After 3 h GD, neuro2a cells were replaced by 20-Methyladenine (3MA); Bafilomycin A1 (BA1) and z-VAD-FMK (z-VAD) were added at final concentrations of 5 mM, 5 nM and 20 uM, respectively. (A) Cell viability was assessed using the MTT assay after treatment of autophagy and pan-caspase inhibitors. MTT mitochondrial reduction was shown as a relative percentage of the MTT values at the indicated time point (3 h GD/48 h GR) from three independent experiments ± SD (<i>*p<0.05</i>, <i>**p<0.001</i>). (B) Changes in protein levels by inhibitors of autophagy or caspase activation were analyzed by immunoblot. Proteins were blotted with anti-LC3, p62, cleaved-caspase 3 and PARP antibodies. Tubulin was used as loading control. (C) Quantification of immunoblot data in (B). The amount of each protein was normalized against the amount of tubulin. Data represent the mean ± SD (<i>*p<0.05,</i> n = 3). (D) Cells transiently expressing GFP-LC3 (green) were treated with 3MA, BA1 or z-VAD for 48 h of GR after 3h GD. Nuclei were stained with DAPI (blue). Scale bar represents 5 <i>µ</i>m. Quantification shown on the right graph represents mean GFP-LC3 puncta per cell (n = 10 cells per condition) from three independent experiments ± SD. The asterisks (*) indicate significant differences in the values.<i>*p<0.05, **p<0.001.</i>