(A) NRG1-ERBB3 signaling is enhanced in cells treated with trametinib. Exponentially growing UM001 cells were treated with vehicle or 100 nM trametinib overnight, followed by treatment with increasing doses of NRG1 for 1 hr. Cells were lysed and phosphorylation of AKT, ERBB3, ERBB2 and ERK1/2 assessed by Western blotting. (B) UM001 cells were treated with vehicle or 100 nM trametinib overnight, followed by treatment 2.5 ng/ml of NRG1 for the indicated time points. Lysates were analyzed as in A. (C) UM003 cells were treated and analyzed as in A. (D) UM001 cells were transfected with 20 nM control siRNA or ERBB2 siRNA. After 72 hr, cells were treated with 100 nM of trametinib overnight, followed by stimulation with 10 ng/ml NRG1 for 1 hr. Knockdown efficiency and phosphorylation of AKT, ERBB3 and ERK1/2 was evaluated by Western blotting.