Figure 1

Synaptic acetylcholinesterase (AChE_S) initiates apoptosis in the nuclear compartment. (a) Time-lapse images showing the alteration of the distribution of the indicated proteins during apoptosis. HeLa cells were transfected with the plasmids expressing the indicated fusion proteins. After 24 h, cells were exposed to 100 μM H₂O₂ and time-lapse images were captured using a Leica AS MDW live cell image acquisition system. See also Supplementary Video S1a. (b) Western blot analysis of fusion protein expression. HeLa cells were transfected with the empty plasmid as a control or the indicated fusion protein expression plasmids. After 24 h, cells were harvested. Whole-cell extracts were prepared, resolved by SDS–polyacrylamide gel electrophoresis, and blotted with anti-GFP antibody (Abmart, M20004). (c) Confocal images showing deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining in HeLa cells transfected with the plasmids expressing the indicated proteins. At 48 h after transfection, TUNEL assays were performed. Cells expressing green fluorescence protein (GFP) alone were used as the empty plasmid control, and those expressing histone H2B were used as the negative control. Scale bars, 15 μm. In the detailed image, the cells are indicated by an arrowhead (right panel; ×2 magnification). (d) DNA ladder assay for apoptosis triggered by the indicated proteins overexpressed in HeLa cells for 48 h. (e) Images showing the GFP-positive HeLa cells. At 18 h after transfection, GFP-positive HeLa cells were sorted by fluorescence-activated cell sorting. At 72 h after sorting, the images were taken using an inverted fluorescence microscope. Scale bar, 30 μM. (f) The viability curves of GFP-positive HeLa cells overexpressing the indicated proteins after sorting. The relative number of cells at the indicated time points was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, with measurement of optical density (OD) at 570 nm. The data are presented as the mean±s.d. of triplicate samples from one experiment that is a representative of three independent experiments. NLS, nuclear localization signal.