FIGURE 6.
Rab32 influences TRAIL-mediated apoptosis onset. A, apoptosis onset upon TRAIL binding depends on Rab32 activity and expression levels. HeLa cells were transfected with plasmids coding for Rab32 and its GDP/GTP binding mutants and with scrambled (scr) or Rab32 siRNA. After 48 h, cells were incubated with 500 ng/ml TRAIL and subsequently analyzed for positive annexin V and propidium iodide signals. The amounts of dead cells were normalized to the vector and scrambled siRNA controls, and results from three independent experiments were graphed. **, p < 0.005 for wild type (wt); *, p < 0.01 for Rab32Q85L and Rab32T39N compared with pcDNA3. B, caspase activation upon TRAIL binding depends on Rab32 activity and expression levels. HeLa cells were transfected as in A. After 48 h, cells were incubated with 500 ng/ml TRAIL and subsequently analyzed by Western blot for caspases 3 and 8. fl, full length; act, active. C, apoptosis onset upon inhibition of kinases. HeLa cells were transfected with plasmids coding for Rab32 and its GDP/GTP-binding mutants and with scrambled or Rab32 siRNA. After 24 h, cells were incubated with 1 μm thapsigargin for 24 h and subsequently analyzed for positive annexin V and propidium iodide signals. The amounts of dead cells were normalized to the vector, and scrambled siRNA controls and results from three independent experiments were graphed. D, apoptosis onset upon staurosporine inhibition of kinases. HeLa cells were transfected with plasmids coding for Rab32 and its GDP/GTP binding mutants and with scrambled or Rab32 siRNA. After 48 h, cells were incubated with 1.2 μm staurosporine for 6 h and subsequently analyzed for positive annexin V and propidium iodide signals. The amounts of dead cells were normalized to the vector and scrambled siRNA controls, and results from three independent experiments were graphed. *, p < 0.01 for Rab32Q85L compared with pcDNA3.