Expression of HHV8 vFLIP protects against growth factor withdrawal–induced apoptosis in TF-1 cells.
(A) TF-1 cells were transduced with an empty retroviral vector or vectors expressing Flag epitope–tagged HHV8 vFLIP K13 and EHV2 vFLIP E8. Expression of the transduced proteins was confirmed by immunoprecipitation with Flag antibody beads (Sigma) followed by Western blot analysis with a rabbit polyclonal antibody against the Flag epitope tag. (B) HHV8 vFLIP K13 protects TF-1 cells against growth factor withdrawal–induced cell death. TF-1 cells expressing an empty vector, vFLIP K13, and vFLIP E8 were deprived of GM-CSF for 48 hours and cell viability measured as described in “Materials and methods.” The values shown are means ± SD of 3 independent experiments performed in triplicate. (C) Photomicrograph demonstrating the protective effect of vFLIP K13 against GM-CSF withdrawal–induced apoptosis. Cells were examined under a phase-contrast microscope and photographed. Original magnification, × 200. (D) Hoechst 33342 staining demonstrating the nuclear morphology of the indicated TF-1 cells grown in the absence and presence of GM-CSF. TF-1 vector and vFLIP-E8 cells demonstrate the classical nuclear features of apoptosis, such as nuclear condensation and fragmentation, which are significantly absent in vFLIP K13–expressing cells. Original magnification, × 200. (E) DNA content analysis demonstrating the appearance of a significant population of cells with sub-G0/G1content among TF-1 vector cells when deprived of GM-CSF for 48 hours that is markedly reduced in vFLIP K13–expressing cells. Cell pellets were fixed in 70% ethanol, and incubated at 4°C overnight. For staining, the cell pellets were resuspended in 0.5 mL of 0.05 mg/mL PI plus 0.2 mg/mL RNase A and incubated at 37°C for 30 minutes. Cell cycle distribution was analyzed using a flow cytometer.
