Figure 1.
Characterization of a fluorescent XIAP fusion protein. (a) The generated XIAP fusion protein (C-XIAP-C). Full-length XIAP was fused at both NH2 and COOH termini with Cerulean fluorescent protein to allow for highly sensitive XIAP detection. (b) Parental MCF-7 cells as well as MCF-7 cells transiently transfected to express Cerulean or C-XIAP-C were probed with antibodies against Cerulean or XIAP. C-XIAP-C was detected at its expected molecular weight of 109 kD. α-Tubulin served as loading control. (c) Comparative quantification of XIAP and C-XIAP-C expression in MCF-7 cells. Densitometry was performed on digitally recorded 12-bit images of immunoblots probed with a XIAP antibody. Data are means+S.E.M. from n=3 detections. (d) Expression of C-XIAP-C does not affect cellular morphology. Cells were imaged 24 h after transfection. Cerulean fluorescence was found throughout the cell, whereas C-XIAP-C was predominantly found in the cytosol. Scale bar=10 μm. (e, f) C-XIAP-C inhibits apoptosis. MCF-7 cells transiently transfected to express Cerulean or C-XIAP-C were exposed to TRAIL/CHX (100 ng/ml plus 1 μg/ml) or STS (1 μM) for the indicated times and analysed by flow cytometry. Apoptosis was measured as the percentage of annexin V-FITC-positive/PI-negative cells. Addition of caspase inhibitor zVAD-fmk (50 μM) indicated that the apoptotic response to TRAIL/CHX or STS was caspase dependent. Data are shown as mean+S.D. from n=3 samples. *Significant differences (P<0.05; one-way ANOVA and subsequent Tukey's test). Experiments were repeated with similar results. (g) C-XIAP-C has an anti-apoptotic potency comparable to native XIAP. MCF-7 cells transiently transfected to express Cerulean, XIAP or C-XIAP-C were exposed to TRAIL/CHX (100 ng/ml plus 1 μg/ml) for 4 h and analysed by flow cytometry. Apoptosis was measured as the percentage of annexin V-FITC-positive/PI-negative cells. Data are shown as mean+S.D. from n=3 independent samples. *Significant induction of apoptosis (P<0.05); NS, not significant (one-way ANOVA and subsequent Tukey's test). Experiment was repeated with similar results. (h) C-XIAP-C interacts with Smac. XIAP-deficient HCT-116 cells transiently transfected to express C-XIAP-C were lysed and immunoprecipitation was performed in native cell extracts with a Smac bait antibody. After western blotting, C-XIAP-C was detected with an antibody directed against XIAP