Research Paper Volume 8, Issue 7 pp 1470—1484

Figure 1. Caloric restriction increases autophagy in rat cortical neurons. Primary rat cortical neurons were exposed to caloric restriction mimic medium (CR), DMEM low glucose, for 6 h. Untreated cells were used as control (Ctrl). (A) LC3B puncta immunoreactivity was assessed by immunocytochemistry, as described in Materials and Methods. Cells were immunolabeled for LC3B (green) and MAP2 (red). Nuclei were stained with Hoechst 33342 (blue). Figures are representative of three independents experiments. Scale bar, 20 μM. (B) Quantification of LC3B puncta immunoreactivity (green) per cell in each condition (>20 cells per group). ***p<0.001, significantly different compared to control, as determined by Student’s t test. (C, D and E) Cells were incubated with chloroquine (ChQ, 100 μM), a lysosomal degradation inhibitor, 30 min before caloric restriction medium for 6 h. Whole cell extracts were assayed for LC3B-II (C), SQSTM1 (D), phospho-MTOR (p-MTOR) (E) and β-tubulin (loading control) immunoreactivity through Western blotting analysis, as described in Materials and Methods. Representative Western blots for each protein are presented above each respective graph. The results represent the mean ± SEM of, at least, five independents experiments, and are expressed as percentage of control. **p<0.01 and ***p<0.001, significantly different compared to control; ^###p<0.001, significantly different from caloric restriction; ^$$$p<0.001, significantly different from chloroquine-treated cells, as determined by ANOVA, followed by Bonferroni’s post test.