Fig. 7. EGF enhances the growth inhibitory effect of trametinib.

a Metastatic MCF10-Ca1a cells were constructed to express a control (TetOn-MT) or EGFR (TetOn-EGFR) encoding vector under the control of doxycycline (Dox) inducible promoter. These cells were pretreated with Dox for 24 h and subsequently stimulated with EGF (50 ng/ml) for 30 min in the presence or absence of trametinib (TRAM). Phosphorylation of EGFR, STAT1, and ERK1/2 was assessed. Expression of total EGFR, STAT1, and ERK1/2 served as loading controls. b MCF10-Ca1a TetOn-EGFR cells were stimulated with the indicated concentrations of Dox for 24 h and assessed for expression of EGFR. β-Tub served as a loading control. c MCF10-Ca1a TetOn-EGFR cells were stimulated with the indicated concentrations of Dox for 10 days in the presence or absence of EGF and trametinib and cell viability was quantified. Chart inset: The resultant P values of ANOVA analyses comparing the indicated treatment groups under control (Dox 0) and Dox conditions. d The MDA-MB-468 cells were stimulated with EGF (50 ng/ml) for 30 min in the presence or absence of trametinib and assessed for phosphorylation of EGFR, STAT1, and ERK1/2. Expression of total EGFR, STAT1, and ERK1/2 served as loading controls. e The MDA-MB-468 cells were grown for a period of 10 days in the presence or absence of EGF (50 ng/ml), IL6 (20 ng/ml), trametinib (5 nM) of the indicated combinations at which point cell viability was quantified. The indicated groups were analyzed by T-test resulting in the indicated P values. Data in panels a, b, and d are representative of three independent analyses, and data in panels c and e are the mean ± SE for three independent experiments completed in triplicate