Figure 2
LiCl rescues GFP-expPABPN (13Ala and 17Ala)-associated cell death. (a) Cell survival determined by live-stage microscopy. C2C12 cells were transfected with a GFP vector, GFP-wtPABPN1-10Ala, GFP-expPABPN1-13Ala, and GFP-expPABPN1-17Ala, treated or not with 2.5 mM LiCl. The cells were counted every 24 h post transfection for 6 days consecutively using the live-stage microscope. The percentage of transfected living cells represents the variation of the amount of transfected living cells at different time points compared with the number of transfected cells obtained on day 1. LiCl rescues GFP-expPABPN1-13Ala and 17Ala-associated cell death (*P<0.001 versus non-treated samples). Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated 3 times. (b) Cell death measured by fluorescent flow cytometry (FFC). Percentage of cell death observed by two-color FFC analysis with 7AAD on day 6 post-treatment with 2.5 mM LiCl. GFP-wtPABPN1-10Ala and GFP were used as controls. Cell death was calculated by dividing the number of 7AAD-stained transfected C2C12 cells over the total number of transfected cells. The experiment was repeated four times. (*P<0.001 versus non-treated samples). (c) A two-color FACS dotplot from a representative experiment in which C2C12 cells were transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl (bottom) compared with non-treated cells (top), stained with 7AAD to label dead cells, and co-sorted for GFP (green) and 7AAD (red) fluorescence. Grid lines were positioned after calibrating the flow cytometry. Upper right quadrants signify 7AAD-labeled dead or dying transfected cells (GFP+7AAD+), i.e., Q2 (underlined values)
