Western blotting analyses and immunoprecipitations. Cells (3 × 105 cells/well) were seeded in six-well plates, grown overnight and then treated with 50 μg/ml HW1 for the indicated periods with or without the JNK inhibitor, SP600125 (10 μM) (Calbiochem). The standard procedures for Western blotting were then done as described previously [13]. For immunoprecipitations, Veliparib (2 × 107 cells/time point) were treated with HW1 (50 μg/ml) for the indicated periods, collected, and lysed, as essentially described before [13]. The cleared cell lysates were then subjected to immunoprecipitations with anti-Beclin-1 rabbit Ab and then agarose-conjugated anti-rabbit Ab (Sigma), followed by western blotting analysis of coimmunoprecipitated Bcl-2 and Bcl-xL.
Results and discussion
Constitutive JNK activation enhanced HW1-mediated cell death
Fig. 1.
Enhanced HW1-mediated cell death by constitutive JNK activation, which was significantly inhibited by the autophagic inhibitor 3-MA. (A) Cell viability of HCT116 cells untransfected or transiently transfected with the plasmid of pcDNA3-Flag-MKK7-JNK1 (expressing constitutively active JNK1) or pcDNA3-Flag-MKK7-JNK1(APF) (expressing phosphorylation-negative JNK1 mutant), which were treated with the indicated concentrations of HW1 for 36 h. (B) Effects of 3-MA on the cell viability of HCT116 cells untransfected or transiently transfected with the plasmid expressing constitutively active JNK1 and p53?/? HCT116 cells, which were pretreated for 1 h with 125 μM 3-MA and further incubated with 50 μg/ml HW1 for 36 h. Error bars indicate the standard deviations for at least triplicated experiments. Significance was determined using the Student’s t-test versus control samples not pretreated with 3-MA (?P
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