Fig. 13
Expression of NHE6∆ES induces apoptotic cell death of primary hippocampal neurons. a Representative confocal images of primary hippocampal neurons transfected with cytosolic GFP alone or GFP co-transfected with either NHE6ChFP-WT or NHE6ChFP-∆ES. Forty-eight h post-transfection, cells were fixed, permeabilized, blocked, and assessed for apoptosis by immunostaining for cleaved caspase-3 (cCASP3, blue). For each transfection condition, an overview is presented of the entire transfected neuron with the GFP, ChFP, and cCASP3 channels merged (middle panels) with higher magnification cut-away images of the area around the cell soma (indicated by the white square) with each channel displayed separately (right panels). For the co-transfected cells, the signals for the ChFP-tagged NHE6 constructs are also shown separately (left panels). As noted in the images, cCASP3 was also detected in nontransfected cells, which could include not only neurons, but also astrocytes and glia. Hence, to estimate the background level of apoptotic cells per field of view, companion cultures in each preparation were fixed, permeabilized and immunolabelled for cCASP3 and stained with propidium iodide to mark the nuclei in order to calculate total cell density/field of view. Under each treatment condition, the average number of cells per field of view ranged from 350 to 400 and the percentage of apoptotic cells per field of view for GFP, GFP + NHE6ChFP-WT or GFP + NHE6ChFP-∆ES was 9.8 % ± 0.7, 9.7 % ± 1.0 and 9.1 % ± 0.8 (mean ± S.E.M.), respectively. Hence, ~10 % of the cells in the background were apoptotic under each condition. b Quantitative representation of the percentages of cCASP3-positive (cCASP3+) neurons of examined GFP or GFP- and NHE6ChFP transfected cells within each condition for four separate experiments. Data are presented as the mean ± S.E.M. (total transfected cells examined ranged from 21–25 cells per condition). Compared to transfection with GFP alone or GFP + NHE6ChFP-WT, significantly more neurons transfected with GFP + NHE6ChFP-∆ES were cCASP3-positive. *: p < 0.01, one-way ANOVA with Tukey post-hoc test. Scale bar: whole cell images, 60 μm; high magnification images, 10 μm