Fig. 5.

HER receptors are activated by MEK inhibitor in MAP3K1; MAP2K4 wild-type cells. a Control and MAP3K1 or MAP2K4 knockout H358 or HCT116 cells were treated with 2 μM selumetinib for 72 h and lysates were western blotted for p-HER3, HER3, p-JUN, JUN, MAP2K4 or MAP3K1, p-ERK, ERK. HSP90 served as a control. b Control and MAP3K1 or MAP2K4 knockout MDA-MB-468 cells were treated with 0.02 μM trametinib for 72 h. The levels of p-HER3, HER3, p-JUN, JUN, p-ERK, ERK, MAP3K1 and MAP2K4 were determined by western blot analysis. c Two breast cancer cell lines of indicated MAP2K4 mutation status were treated with 2 μM selumetinib for 72 h and lysates were subjected to phospho-RTK activation analysis. Dots labeled 1-4 represent duplicate blots of p-EGFR, p-HER2, p-HER3 and p-HER4, respectively. d MDA-MB-468 cells were treated with selumetinib for 72 h, then RNA was extracted and qRT-PCR analysis was performed for HER receptor transcripts. e Two individual shRNAs targeting JUN were introduced into MDA-MB-468 cells by lentiviral transduction. Ctrl and JUN knockdown cells were treated with 2 μM selumetinib for 72 h. The levels of phospho-HER1-4 and HER1-4 receptors, p-JUN, JUN, p-ERK and ERK were determined by western blot analysis. HSP90 served as a loading control. f MDA-MB-468 cells were cultured for two weeks in medium containing increasing concentration of selumetinib alone, dacomitinib (8 nM) alone, or combination of selumetinib and dacomitinib. After this, cells were fixed and stained