FIG. 8.
Prolonged JNK activation affects the susceptibility of cells to TNF-α-induced apoptosis. (A) TNF-α-sensitive LM cells were treated with TNF-α (20 ng/ml) and harvested at the time points indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (B) LM cells were cotransfected with expression vectors encoding M2-JNK (1.5 μg) and either the JNKK2(K149M) mutant or an empty vector (6.0 μg of each). Cells were treated with or without TNF-α (20 ng/ml) for 15 min. The activity of M2-JNK and the expression of transfected constructs were determined as previously described (49). (C) LM cells were cotransfected with expression vectors encoding GFP (1.5 μg) and either the JNKK2(K149M) mutant or an empty vector (6.0 μg of each). Cells were treated with or without TNF-α (20 ng/ml) for 8 h, stained with Hoechst dye, and visualized by fluorescence microscopy. The death rate of transfected (GFP-positive) cells was calculated by counting six randomly selected areas (total, 30 to 40 cells per area). Results are presented as means ± standard errors and represent two individual experiments. (D) TNF-α-insensitive HT-1080 cells were treated with TNF-α (20 ng/ml) for various times as indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (E) HT-1080 cells were infected with Ad/IκBα(A32/36) (MOI, 500). After 24 h, cells were treated with TNF-α (20 ng/ml) for various times as indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (F) HT-1080 cells infected with Ad/IκBα(A32/36) (MOI, 500) were stimulated with TNF-α (20 ng/ml). After 30 min, cells were treated with SP600125 (20 μM) and harvested at the time points indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (G) HT-1080 cells were infected with Ad/IκBα(A32/36) or Ad/GFP (MOI, 500) and treated with TNF-α (20 ng/ml). After 30 min, cells were treated with or without SP600125 (20 μM) and cell death was calculated 9 h later as described in the legend to Fig. 2C. The results shown represent three separate experiments.