Overexpression of SOK1 induces apoptotic cell death. A,
overexpression of SOK1 induces cell death. HEK293, SaOS2, or COS7 cells were
transfected with pCMV5-SOK1 (SOK1) or pCMV5 plasmid (vector), and HeLa cells
were infected with either an adenovirus expressing GFP (Ad-GFP), or an
adenovirus expressing both SOK1 and GFP (Ad-SOK1). The percentage of dead
cells was determined by trypan blue exclusion 72 h after transfection of
HEK293, SaOS2, and COS7 cells and 48 h after infection of HeLa cells. Shown is
the mean and S.D. of four independent experiments. *, p
< 0.05 versus empty control. B, the levels of
overexpressed SOK1 correlate with its cell death-inducing capacity. HEK293
cells were transfected with increasing amounts (0, 1.25, 2.5, 3.75, or 5
μg) of pCMV5-M2SOK1 (SOK1). The percentage of cell death was determined by
trypan blue exclusion 72 h after transfection. GAPDH is shown as a loading
control. C, overexpression of SOK1 induces chromatin condensation and
membrane blebbing. HeLa cells were infected with Ad-SOK1, which also expresses
GFP, and visualized in vivo. GFP was used to score for successfully
infected cells (left panel). Chromatin condensation was visualized by
Hoechst 33342 (central panel), and cell morphology was evaluated
using Hoffman interference microscopy (right panel). GFP-positive
cells with apoptotic features are marked with arrows. D, SOK1 induces
cytochrome c release to the cytoplasm. HEK293 cells were transfected
with control or a SOK-1 expressing plasmid and either incubated with 500
μm H2O2 or left untreated. Cytoplasmic
cytochrome c (cyt. c) was determined by Western blot.
E, SOK1-induced cell death is caspase-dependent. HEK293 cells were
transfected with empty plasmid (pCMV5) or pCMV5-SOK1 (SOK1) and treated with a
caspase inhibitor (z-VAD-fmk or Ac-DEVD-CHO) or vehicle. The percentage of
non-viable cells was determined by trypan blue exclusion. Shown is the mean
and S.D. of four independent experiments. *, p < 0.05
for SOK1 + caspase inhibitors versus SOK1 alone. F, PARP is
cleaved during SOK1-induced cell death. Extracts were prepared from HeLa cells
infected with control or SOK1-expressing adenovirus for the times indicated.
Western blots were performed for SOK1 and PARP. The arrow marks the
predicted position of caspase-cleaved PARP.