Fig. 8

MEKi withdrawal from cells with KRAS^G13D amplification/upregulation induces a ZEB1-dependent EMT. a HCT116 and H6244-R cells were treated with 2 μM selumetinib (H6244-R + Sel) or DMSO only (HCT116, H6244-R − Sel) and imaged by brightfield phase contrast microscopy after 9 days. Scale bars indicate 100 µm. b HCT116 and H6244-R cells were treated with 2 μM selumetinib (H6244-R + Sel) or DMSO only (HCT116, H6244-R − Sel) for 9 days and stained for CDH1 (red) or VIM (green) and nuclei (blue). Scale bars indicate 50 µm (upper panels) and 10 µm (lower panels). c HCT116 and H6244-R cells were treated with 2 μM selumetinib (H6244-R + Sel) or DMSO only (HCT116, H6244-R − Sel) for 9 days and relative expression of the indicated mRNAs (normalized to B2M) determined by RT-qPCR. Results are mean ± SD of at least three independent experiments. P < 0.001 (***), P < 0.01 (**), P < 0.05 (*), P > 0.05 (ns) as determined by one-way ANOVA with Tukey’s multiple comparisons test. d HCT116 and H6244-R cells were treated with 2 μM selumetinib ( + Sel) or DMSO only (HCT116, − Sel) for the indicated times. e LoVo and L6244-R cells were treated with 4 μM selumetinib ( + Sel) or DMSO only (LoVo, − Sel) for the indicated times. f HCT116 and H6244-R cells were either left untransfected (UT), transfected with non-targeting (NT) siRNA or transfected with SNAI1-, SNAI2-, or ZEB1-specific siRNA as indicated. Twenty-four hours later, cells were treated with 2 μM selumetinib (+) or DMSO only (−) for 48 h. g LoVo and L6244-R cells were either left untransfected (UT), transfected with non-targeting (NT) siRNA or transfected with SNAI1- and/or ZEB1-specific siRNAs as indicated. Twenty-four hours later cells were treated with 4 μM selumetinib (+) or DMSO only (−) for 48 h. d–g Lysates were western blotted with the indicated antibodies and results are representative of at least two experiments giving equivalent results. A549 cells were used for positive control (C), except for SNAI2 (SW620 cells)