Fig. 2.

Cd triggers accumulation of autophagosomes contributing to neuronal cell death. PC12 cells and primary neurons, or PC12 cells and primary neurons infected with Ad-GFP-LC3, were pretreated with/without 3-MA (4 mM) for 1 h and then exposed to Cd (10 μM) for 8 h (for Western blotting), 12 h (for MDC staining and GFP-LC3 assay) and 24 h (for DAPI and TUNEL staining). (A) The fluorescence intensity for MDC-labeled vacuoles in the cells was quantified. (B) The ratio of cells with large LC3 punctate structures was counted and calculated. (C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (D) The blots for LC3-II, p62, cleaved-caspase-3, and cleaved-PARP were semi-quantified. (E) Apoptotic cells were evaluated by nuclear fragmentation and condensation (arrows) using DAPI staining (upper panel) and concurrently by in situ detection of fragmented DNA (in green) using TUNEL staining (lower panel). Scale bar: 20 μm. (F and G) The percentages of cells with fragmented nuclei and the number of TUNEL-positive cells were quantified. For (A), (B), (D), (F) and (G), all data were expressed as means ± SE (n = 3–5). *P < 0.05, difference with control group; ^#P < 0.05, difference with 10 μM Cd group.