Figure 6.

wt Scotin protein has an intrinsic proapoptotic activity. (A) Schematic representation of the Scotin mutants. (B) Scotin mutants deleted of the NH₂ terminus are located in the ER. H1299 cells were transfected with 0.5 μg of AdScotin-FLAG (a), or SVScotin-FLAG (b), or AdΔCys (c, or AdΔN (d), or SVΔpro (e). Cells were stained with anti-FLAG (M2) mouse mAb followed by anti–mouse antibody conjugated to FITC. Bars, 5 μm. (C) wt Scotin protein induces apoptosis after transfection. The DNA content of each transfected population was determined by flow cytometry analysis. The percentage of sub-G1 DNA content represents the percentage of apoptotic cells 48 h after transfection. H1299 cells transfected with the following different expression vectors: 5 μg/ml AdCAT, or 10 μg/ml SVScotin, or 5 μg/ml AdScotin, or 2 μg/ml SVp53, or 5 μg/ml AdΔCys, or 5 μg/ml AdΔN. Histogram represents the average of at least three independent transfections. SDs are reported as error bars. (D) Scotin-mediated apoptosis is inhibited by caspase inhibitor. The DNA content of each transfected population was determined by flow cytometry analysis. H1299 cells transfected with 5 μg/ml of AdCAT, 5 μg/ml of SVScotin-FLAG, or 5 μg/ml of AdScotin-FLAG were incubated in presence or absence of the caspase inhibitor Z-VAD-FMK. Histogram represents the average of at least three independent transfections. SDs are reported as error bars. (E) Scotin induces caspase-3 activation. The caspase-3 activity was determined using the Colorimetric Caspase-3 Cellular Activity Assay kit. The H1299 cells were transfected with 5 μg/ml of AdCAT, 5 μg/ml of AdScotin-FLAG, or 2 μg/ml of SVp53 expression vectors. As indicated, cells were incubated in the presence or absence of the caspase inhibitor Z-VAD-FMK. Histogram represents the average of three independent transfections. SDs are reported as error bars.