Apoptotic assays of transfected NSC34 and Neuro2a cells. (A) Comparison of apoptotic death induced by exogenous WT and MT hTDP-43. Cell death was assayed by measurement of the activities of caspase 3/ 7 at 24 hr and 72 hr post-transfection with the expression plasmids (5 ug/ 106 cells). Mock, cells without transfection; C, cells transfected with the pEF vector; Stau. 5 uM, cells treated with 5 uM of staurosporine for 6 hr to induce apoptosis. The folds of the caspase activities relative to that of the Mock sample were calculated and shown. Note the significant increase of the caspase 3/ 7 activities in NSC34 cells (left panel), but not Neuro2a cells (right panel), induced by the two MT hTDP-43 forms at 72 hr post-transfection. (B) Comparison of plasmid dose-dependent apoptotic death induced by exogenous WT and MT hTDP-43. Apoptotic cell death was determined by immunofluorescence staining of cleaved caspase 3. The extents of apoptotic cell death of NSC34 cells and Neuro2a cells at 72 hr post-transfection with different amounts of the expression plasmids were assayed by immunofluorescence staining with the antibodies 2E2D3 and Ac-cap 3. Means of three independent experiments (S.D.) are plotted in the upper 2 panels, with the % of hTDP-43-positive cells that are also Ac-cap 3-positive as a function of the doses of transfection. Approximately 1% of cells transfected with the pEF vector were Ac-cap3 positive (◆ in the two plots). Representative photographs are shown below the plots, with the apoptotic nuclei/ cleaved caspase 3-positive cells indicated by the arrowheads. For both the NSC34 and Neuro2a sets, two Ac-cap 3-positive cells (the boxed areas) are magnified for better visualization. Scale bar, 10 μm. *, p< 0.05; **, p< 0.01; ***, p< 0.001.