Synergy between AR and MEK inhibitors in trastuzumab-resistant cells. (A) MTT assay was used to measure cell viability in MDA-MB-453 cell line following trastuzumab treatment at 10 to 80 μg/ml concentrations. CTL: control. *P < 0.01 for trastuzumab groups vs. control. Error bars: ± 2 SEM. (B) Cell viability in HCC-1954 cell line following trastuzumab treatment as described in Figure 8A. (C) Cell viability in trastuzumab-resistant MDA-MB-453 (R) compared to the untreated control and control line treated with trastuzumab at 20 μg/ml. *P < 0.01 for R vs. treated control cells. (D) Combination indices (CI) for flutamide (FLU) and CI-1040 combination therapy in MDA-MB-453-R line using MTT assay. Therapies were carried out with flutamide at 5 and 10 μM with each concentration of CI-1040 at 5 and 10 μM (CI-5 and CL-10). The concentrations of fluatmide and CI-1040 monotherapies with an effect similar to that of each combination therapy are depicted. Error bars: ± 2 SEM. (E) Western blot analysis was used to measure the phosphorylated (ph) and total ERK levels in MDA-MB-453-R, control MDA-MB-453 and control MDA-MB-453 treated with trastuzumab at 20 μg/ml concentration (CTL + Tras). Fold changes in band densities (RR) were measured relative to the control. (F) Western blot analysis was used to measure the phosphorylated and total ERK levels in MDA-MB-453-R line following combination therapies with CI-1040 (5 μM)/flutamide (5 μM) and CI-1040 (5 μM)/flutamide (10 μM). RR values were measured relative to the untreated MDA-MB-453-R line.