Fig. 4.

MEK inhibitor activates JNK-JUN signaling through suppression of DUSP4. a Five MAP3K1/MAP2K4 wild-type cell lines were treated with 100 nM thapsigargin for 1 h or 2 μM selumetinib for 6 h, the levels of p-JUN and JUN were determined by western blot analysis. HSP90 served as a loading control. b Control and MAP2K4 knockout H358, LoVo or HCT116 cells were treated with 2 μM selumetinib for 72 h and lysates were western blotted for DUSP4, DUSP16, p-JUN, JUN, p-ERK, ERK. HSP90 served as a control. c Two individual shRNAs targeting DUSP4 or a DUSP4 expression vector (DUSP4 OE) were introduced into H358, LoVo or HCT116 cells by lentiviral transduction. Lysates of control and DUSP4 knockdown or overexpression H358, LoVo or HCT116 cells were western blotted for DUSP4, p-JNK, JNK, p-JUN, JUN, p-ERK, ERK. HSP90 served as a control. d Control and DUSP4 overexpression H358, LoVo or HCT116 cells were cultured with normal medium or medium containing 0.25 μM (LoVo) or 4 μM (H358 and HCT116) selumetinib. Percent confluence over time was monitored using IncuCyte