EGFR and EpCAM levels are molecular determinants of EMT induction, ERK activation, and migration.

(A) Kyse30 cells were transfected with EGFR-specific siRNAs (pool of n = 4 siRNAs) (EGFR KD), siRNA controls (siRNA ctrl), EpCAM-specific shRNA (EpCAM KD) [54], control shRNA (shRNA ctrl), and a combination of EGFR siRNA and EpCAM shRNA (double KD). Expression of EGFR and EpCAM was assessed by immunoblotting with specific antibodies. Equal loading was confirmed by detecting actin levels. Clinical quadrants’ equivalents are indicated. Shown are representative results from n = 3 independent experiments. (B) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGFlow (1.8 nM) and EGFhigh (9 nM). Cell morphology was assessed after 72 hr. Shown are representative images from n = 3 independent experiments. (C) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGFlow (1.8 nM) for the indicated time points, and ERK1/2 phosphorylation was assessed by immunoblotting. Levels of ERK1/2 and actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (D) Quadrant 1 to 4 equivalents of Kyse30 cell variants were either kept untreated (control) or were treated with EGFlow (1.8 nM). After 72 hr, mRNA transcript levels of Slug were assessed by qRT-PCR with specific primers. Shown are means with SDs from n = 2–3 independent experiments performed in triplicates. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p-values S1 Data. (E) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGFhigh (9 nM) and subjected to a scratch assay. Relative migration was quantified from representative micrographs of each cell line. Shown are means with SDs from n = 3 independent experiments. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p-value S1 Data. EGF, epidermal growth factor; EGFR, EGF receptor; EpCAM, epithelial cell adhesion molecule; EMT, epithelial-mesenchymal transition; ERK, extracellular signal–regulated kinase; KD, knockdown; pERK1/2, phosphorylated ERK1/2; qRT-PCR, quantitative real-time PCR; SD, standard deviation; shRNA, short hairpin RNA; siRNA, small interfering RNA; WT, wild type.