Figure 5.

SKIP re-expression is associated with increased apoptotic signals. A, SKIP transfection did not affect growth of CTS and K562 cells under normal conditions. B, after 24 h serum starvation there were significantly less viable SKIP-transfected cells than vector-transfected cells for both K562 and CTS cells (n = 3). The SK1 inhibitor 5C reversed the pro-apoptotic effect of serum starvation in SKIP-transfected cells. C, Western blots showing higher expression of cleaved PARP (89-kDa fragment) in SKIP-transfected leukemia cell lines compared with vector-alone after 24 h of serum starvation. D, flow cytometry plots showing staining of cells with DAPI and Annexin-V (Annexin-V binds apoptotic cells). In the top left panel is the negative control for Annexin V. In the top right panel is the positive control (irradiated cells) with a high proportion of apoptotic cells (52%). In the bottom panels are vector- and SKIP-transfected CTS cells showing 3.2 and 10.9% apoptotic cells, respectively. E, a higher percentage of cells were apoptotic (positive for both Annexin V and DAPI stains) in SKIP-transfected leukemia cell lines exposed to 24 h of serum starvation compared with vector-alone cells (n = 3). The proapoptotic effect of SKIP transfection was reversed by the SK1 inhibitor 5C in the context of serum starvation. F, SKIP-transfected CTS cells were more sensitive to ara-c chemotherapy than vector-transfected cells (n = 3). The SK1 inhibitor 5C did not reverse the chemosensitivity to ara-c. * = p < 0.05 as measured by unpaired t test.