Intracellular formation of autophagolysosomes following rapamycin treatment in N2a-FK cells.

(A) To determine autophagic activity in 1 μM rapamycin- or 1 μM rapamycin and 10 mM 3MA-treated N2a-FK cells, autophagosomes were detected in transfectants expressing the EGFP-fused LC3 gene (inserted into plasmid pcDNA 3.1) and the morphological changes in LC3-positive granular vesicles were followed. (B) Autophagolysosomes in cells treated with 1 μM rapamycin for 24 h were visualized using 0.1 mM of monodansylcadaverine (MDC) for 30 min (left, three panels per group). To inhibit rapamycin-induced autophagy, N2a-FK cells were co-treated with 10 mM 3MA. Scale-bars represent 10 μm. To quantify the average number of MDC-labeled autolysosomes in a single cell, the vesicles in the treated cells were shown as a graph represented by the mean ± SD of three independent experiments (right). **p < 0.01 (one-way ANOVA followed by Tukey's test). (C) N2a-FK cells treated with 1 μM rapamycin for 24 h were pre-treated with 3 M guanidine thiocyanate prior to the antibody reaction. PrPSc in cells was detected using the SAF61 antibody (green) and N2a-58 cells were stained as a negative control. Cell nuclei were counterstained with DAPI (blue). The cells were visualized by confocal laser scanning microscopy. Differential interference contrast (DIC) images were obtained to confirm the consistency of the experimental condition. Scale-bars represent 10 μm.