Figure 3.

Functionality of mutant FAS variants. (a) Set-up of the effective dose for CH11-mediated cell death induction, showing the normalized cell death of JK cells treated with increasing doses of CH11. Normalization was made as follows: first, all data were divided by the result for PI staining of non-stimulated cells (0 ng/ml CH11), thus setting the latter as 1. Second, the results for PI staining of irrelevant IgM-treated cells were subtracted from their corresponding CH11-treated cells. (b) FAS-mediated cell death induction in JK cells transfected with wild-type or mutant FAS variant pKEX constructs. Histogram represents the mean and S.D. from at least three independent experiments of 50 and 100 ng/ml of irrelevant IgM and CH11 induction. The percentage of PI-positive cells is showed, previously subtracting from all data the result for non-stimulated cells. Dotted lines represent the result for % PI-positive cells treated with 50 ng/ml (black line) or 100 ng/ml (gray line) CH11 in JK cells not transfected. Significance is indicated by the corresponding P-value. n.s., not significant. (c) Representative experiment of flow cytometry showing PI positivity 24 h after 100 ng/ml CH11-triggering by endogenous FAS (dashed line), or exogenous wild-type (shaded) or mutant FAS variants (solid line). (d) FAS-mediated caspase 8 activation in JK cells transfected with wild-type or mutant FAS variants pKEX constructs, 8 h after 100 ng/ml CH11-triggering. Numbers indicate fluorescence intensity corresponding to the cleaved caspase 8 staining