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. 2008 Jun 6;283(23):16248–16258. doi: 10.1074/jbc.M709724200

FIGURE 1.

FIGURE 1.

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.