Figure 3

Pristimerin inhibits TNFα-induced degradation of IκBα and translocation of p65. KBM5 or KBM5-T315I cells were pretreated with or without 200 nM pristimerin for 6 hours, then treated with TNFα (0.1 nM) at the indicated times; cytoplasmic (A) and nuclear (B) extracts were examined by Western blot analysis with specific antibodies against total and phosphorylated IκBα, IKK and p65, respectively. The same membranes were stripped and reprobed with actin or PCNA. (C) Dose- and time-dependent effect of pristimerin. KBM5 cells were preincubated with the indicated concentrations of pristimerin for 6 hours (left) or 200 nM pristimerin for various durations (right); then treated with TNFα (1 nM) for 5 minutes; cytoplasmic and nuclear extracts were examined by Western blot analysis. (D) Immunofluorescence staining analysis of p65. KBM5 cells were preincubated with 200 nM pristimerin for 6 hours, and TNFα (1 nM) for 5 minutes, fixed in 3% paraformaldehyde, then underwent immunofluorescence analysis against p65 and FITC-conjugated secondary antibody. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (E) Pristimerin prevented the phosphorylation of IκBα in the presence of proteosome inhibitor. KBM5 cells were treated with 500 nM pristimerin in the absence or presence of MG-132 (0.5 μM) for 6 hours, then treated with TNFα (0.1 nM) for 30 minutes. Cytoplasmic extracts of cells underwent immunoblotting with phosphopecific anti-IκBα. (F) Pristimerin diminishes the expression of NF-κB-regulated proteins involved in survival. Western blot analysis of K562 cells pretreated with 400 nM pristimerin for 6 hours, then stimulated with TNFα (1 nM) for different times.