Figure 5
ABT-263 enhances ERK- and JNK-mediated Mcl-1Thr163phosphorylation and protein stabilization. (A) HCC cells were treated with 5 μM ABT-263 for 18 h, and then the phosphorylation of ERK1/2, JNK and mTOR were detected by Western blot, taking α-tubulin as a loading control. (B) After pretreated with 10 μM ERK1/2 inhibitor U0126, 10 μM JNK inhibitor SP600125 (SP), or 2 μM mTOR inhibitor rapamycin for 2 h, the HCC cells were treated with 5 μM ABT-263 for another 18 h. Then the level of Mcl-1 protein was analyzed by Western blot. (C) After pretreated with 10 μM U0126 or 10 μM SP600125 for 2 h, the HCC cells were treated with 5 μM ABT-263 for 24 h, then the cells were stained with annexin V-FITC/PI and analyzed by flow cytometry. (D) After pretreated with 10 μM U0126 or 10 μM SP600125 for 2 h, the HCC cells were treated with 5 μM ABT-263 for another 24 h. Then the total cell death was calculated by trypan blue exclusion assay. Data were expressed as mean ± SD from three independent experiments. (*: P < 0.05, ***: P < 0.0001). (E, F) After pretreated with 10 μM U0126 or 10 μM SP600125 for 2 h, the HCC cells were treated with 5 μM ABT-263 for another 18 h. Then PARP and cleaved PARP, p-ERK, p-JNK, Mcl-1 and p-Mcl-1(Thr163) were analyzed by Western blot.