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. 1997 Apr 1;94(7):3302–3307. doi: 10.1073/pnas.94.7.3302

Figure 5.

Figure 5

Effect of an ICE-inhibitor and Bcl-2 on Fas-induced cell death and JNK activation. (A) SHEP cells were incubated for 1 hr with or without the ICE-like protease inhibitor peptide Z-VAD-fmk (50 μM) (Enzyme Systems Products). Anti-Fas (1 μg/ml) was added to the wells for 8 hr. Cell death was determined after 8 hr by a crystal violet staining/spectrophotometric assay. The mean percentage of cell death is shown. Data represent the average of two different experiments performed in triplicate. Error bars indicate the SD. JNK protein kinase activities were measured 4 hr after addition of the anti-Fas. The kinase activities are expressed relative to cells incubated without anti-Fas. Data represent the average of two different experiments. Error bars indicate the SD. (B) Cells were transfected by electroporation using the vector pCEP9 containing the mouse Bcl-2 cDNA in the sense orientation: (Bcl-2). Bcl-2 (p26) protein levels in control SHEP and Bcl-2 cells were determined by immunoblotting. Cells were plated in 96-well plates and the anti-Fas antibody (100 ng/ml) was added for 24 hr. Cell survival was determined after 24 hr by a crystal violet staining/spectrophotometric assay. The mean percentage of cells resistant to cell death after 24 hr is shown. Data represent the average of triplicate cultures from 10 independent experiments. Error bars indicate the SD. SHEP and Bcl-2 cells were plated in six-well plates. Anti-Fas antibody (1 μg/ml) was added to the cells for 4 hr and JNK protein kinase activity was measured. The kinase activities are expressed relative to cells incubated without anti-Fas. Data represent the average from two different experiments performed in duplicate. Error bars indicate the SD. (C) Cell survival and JNK protein kinase assays in parental and Bcl-2 antisense-transfected HCT-15 cells. HCT-15 cells were transfected by electroporation using the vector pCEP9 alone (HCT-15) or containing the mouse Bcl-2 cDNA in the antisense orientation (AS). Data represent the average of triplicate cultures from three independent experiments. Error bars indicate the SD. One kinase assay is shown. The experiment was repeated twice and despite different JNK activity levels, the increase in JNK activation in AS as compared with HCT-15 was reproducibly observed.