FIGURE 3.
MARCH overexpression inhibits apoptosis induction by TRAIL. A, MCF-7Casp-3 cells were transfected to express either GFP only, or GFP-tagged MARCH-1, or -8. At 24 h after transfection, cells were stimulated with IZ-TRAIL for 5 h and caspase-3 cleavage was determined by flow cytometry in the GFP positive cell populations. B, as in A, with the following adaptations. Cells were transfected with WT MARCH-1 or -8, or with the MARCH-8 RING mutant described in the legend to Fig. 2. Cells were stimulated with TRAIL for 14 h, and cell death was read out by propidium iodide (PI) uptake. Data in A and B represent mean ± S.D. of values from 3 to 4 independent experiments. The percentage of cells with cleaved caspase-3 or PI uptake in the untreated control samples was subtracted. Asterisks indicate statistically significant differences between MARCH.GFP-transfected and GFP only-transfected control cells at the indicated concentration of TRAIL (Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001).