Figure 6

Endogenous synaptic acetylcholinesterase (AChE_S) participates in DNA cleavage via aa 32–138 during apoptosis. (a) Immunoblot analysis of the apoptosis induced by mitomycin C (MMC) in mouse embryonic fibroblasts (MEFs). The cells were treated with MMC (40 μM) for 36 h prior to western blot analysis to detect cleaved poly(adenosine diphosphate ribose) polymerase (PARP) (a biomarker of apoptosis) using PARP antibody (Cell Signaling Technology, #9545). (b) Identification of AChE knockout embryonic mice by reverse transcription-PCR. (c) Confocal images showing DNA cleavage in apoptotic AChE^+/+ or AChE^−/− MEFs. Cells were seeded (5×10⁴) on coverslips in a 24-well plate. Upper panels: 48 h later, the cells were treated with 40 μM MMC (Sigma-Aldrich) for 36 h and the TUNEL assay was performed. Lower panels: at 4 h after seeding, the cells were infected with lentiviruses encoding the indicated proteins, where ‘wt’ indicates wt-AChE_S–GFP and ‘Δ’ indicates AChE_S Δ (aa 32–138)-GFP. After a further 44 h, the infected cells were exposed to 40 μM MMC for 36 h. Then, the TUNEL assay was performed and analyzed by confocal microscopy. Scale bar, 20 μm. (d) Average intensity of TUNEL staining in MEFs was quantified by Leica Confocal Software. Fifty cells were randomly selected and analyzed in each experimental group in c. Values represent the mean±s.d. **P<0.01, ***P<0.001, ^#P>0.05 vs AChE^−/− MEFs without virus infection. Two-tailed Student’s t-test. The data shown are representative of two independent experiments. DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescence protein.