Figure 4

Decreasing Bcr-Abl and its downstream signaling, and inhibiting NF-κB may be independent mechanisms of actions of pristimerin. (A) Dose-dependent downregulation of Bcr-Abl protein by pristimerin. CML cells were exposed to pristimerin for 24 hours, whole cell lysates were subjected to immunoblotting analysis. (B and C) CML cells were treated with 600 nM pristimerin for different durations, downstream signaling molecules of Bcr-Abl (B) and proteins of apoptosis (C) were analyzed by Western blotting. (D) K562 cells were pretreated with MG-132 (0.5 μM) for 2 hours, then exposed to pristimerin for another 24 hours. Immunoblots were shown. (E) Pristimerin inhibits Bcr-Abl mRNA levels as measured by RT-qPCR. KBM5 cells were exposed to escalating concentrations of pristimerin for 24 hours (left top and middle) or 600 nM for various durations (right top and middle). The Bcr-Abl mRNA expression relative to the control was calculated by dividing the comparative expression levels. Colomns, mean;bars, 95% confidence intervals, n = 3. Similar data for Sirt1 (an unrelated control gene) were also shown (bottom). **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post-hoc comparisons, Tukey's test. (F) KBM5 cells were pretreated with 1.0-3.0 μM imatinib for 1 hour, the cells were then stimulated with TNFα (0.1 nM, 5 minutes), NF-κB activation was analyzed with Western blotting. (G) K562 cells were transfected with siRNA duplexes against either human p65, PDGFRα or control siRNA, respectively. Twenty-four hours later, the cells were analyzed by Western blotting. (H) A proposed model to delineate the actions of pristimerin.