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. 2016 Oct 7;6:34933. doi: 10.1038/srep34933

Figure 1. MEK/ERK signaling plays a more important role thanPI3K/AKT signaling in mediating PAK1-mediated cisplatin resistance.

Figure 1

(a) Six lung cancer cell types were treated with four concentrations of cisplatin and the dose response curves were used to calculate the 50% inhibition concentration (IC50) for cisplatin. The expression of PAK1 and its active forms (pS144-PAK1 and pT423-PAK1) in each lung cancer cell type were examined by western blotting. (b) Increasing amounts of PAK1 knockdown plasmid were transfected into high-PAK1 expressing (H441 and H23) cell lines. Alternatively, increasing amounts of expression plasmid were transfected into low PAK1 expressing (H358 and H1355) cell lines. The total amount of transfected DNA was kept constant by adding the control vector. After 48 hr, cell lysates were harvested and evaluated by Western blotting for levels of PAK1 and β-actin protein. β-actin was used as a protein loading control. NC: non-specific shRNA control. VC: Vector control. PAK1-knockdown or PAK1-overexpressing lung cancer cells were treated with four doses of cisplatin and the dose response curves were used to calculate the 50% inhibition concentration (IC50). (c) PAK1-overexpressing H358 and H1355 cells were treated for 5 h with inhibitors of PI3K/AKT (10 μM perifosine) and ERK (10 μM AZD6244). The inhibitors were then removed and the cells were treated with 25 μM cisplatin for an additional 48 h. Cell viability was evaluated with the MTT assay. (d) H23 and PAK1-overexpressing H1355 cells were transfected with β-catenin overexpression plasmid for 24 h. The cells were then treated with AZD6244 for 5 h, the inhibitor was removed, and the cells were treated with 25 μM cisplatin for an additional 48 h. Cell viability was evaluated with the MTT assay. (e) H23 and PAK1-overexpressing H1355 cells were treated with AZD6244 for 5 h, followed by treatment with MG132 for an additional 5 h, and then the cell lysates were evaluated for protein expression by western blotting. All experiments were performed three independent times. The mean values and the standard deviations are indicated as columns with error bars. The samples were derived from the same experiment and gels/blots were processed in parallel. Full-length blots are presented in Supplementary Figures S2 and S3.