Figure 7.
KZ-02 Inhibits Pim-1 Function by Promoting Its Proteasomal Degradation
(A) KZ-02 treatment decreases Pim-1 protein levels. Colo205 and HT29 cells were treated with KZ-02 (10 nmol/L) for 2 h, and Pim-1 protein levels were then detected by western blot analysis.
(B) Both KZ-02 and AZD6244 increase Pim-1 mRNA levels. Tumor cells were treated with KZ-02 (10 nmol/L) or AZD6244 (100 nmol/L) for 2 h, and Pim-1 mRNA levels were then analyzed by real-time PCR. Similar experiment in HT29 cells was shown in Figure S2A.
(C) Tumor cells were treated with either KZ-02 (10 nmol/L) or AZD6244 (100 nmol/L) for 2 h with or without addition of the proteasome inhibitor MG132 (10 μg/mL) for 30 min, and Pim-1 expression was then detected by western blot analysis. Similar experiment in HT29 cells was shown in Figure S2B.
(D) KZ-02 treatment inhibits Pim-1 and its downstream effectors, whereas AZD6244 increases their activation in Colo205 cells. Similar experiment in HT29 cells was shown in Figure S2D.
(E) Tumor cells were treated with various concentrations of KZ-02, AZD6244+Pim-1 1 (1:1), or AZD6244+Pim-1 siRNA (8 nmol/L) as indicated for 24, 48, and 72 h. MTT assay was then used to characterize the inhibition of tumor cell proliferation. Similar experiment in HT29 cells was shown in Figure S2E.
(F) IC50 values were calculated for data in (D) and the ratios of AZD6244+Pim-1 1 to KZ-02 IC50 were shown. Pim-1 knockdown efficiency was confirmed in Figure S2C. Error bars are based on the standard deviations of triplicate samples.
Data are represented as mean ± SD. Comparisons of two groups are performed with Student's t test. ∗∗∗p < 0.001.