TGF-β and EGFR pathways cooperate to induce stemness in GIF-14 cells.

<p>(A) Changes in the expression of stemness- and EMT/mesenchymal-associated marker genes in response to TGF-β1 and EGF. GIF-14 cells were treated with murine EGF (10 ng/ml) or TGF-β1 (2.5 ng/ml) or in combination for 15 h. Quantitative PCR measurements of gene expression levels are normalized against <i>Gapdh</i> levels, and expressed relative to the control sample (means ± SEM, n = 4). Student's t-tests are performed in which single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively and n.s represents not significant (Black bracket: TGF-β1 responsiveness; Red bracket: cooperative induction by TGF-β1 and EGF). (B) Cooperative induction of stemness by EGF and TGF-β1. GIF-14 cells were pretreated with TGF-β1 (2.5 ng/ml) for varying periods at 15 h, 24 h, 48 h and 72 h before the addition of murine EGF (10 ng/ml) for another 15 h. Changes in the mRNA levels of stemness marker <i>Hmga2</i>, regulators of EGF signaling <i>EGFR</i> and <i>Lrig1</i>, EMT markers <i>Snai1</i> and <i>fibronectin1</i> (<i>Fn1</i>) were determined by qRT-PCR. The values are normalized against those of <i>Gapdh</i> and are expressed relative to that of the control (means ± SEM, n = 3). Student's t-tests are performed where indicated. Single and double asterisks represent <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively while n.s denotes not significant (Black bracket: TGF-β1 responsiveness; Blue bracket: EGF responsiveness; Red bracket: cooperative induction by TGF-β1 and EGF). (C) TGF-β1 induction of <i>Hmga2</i> is abrogated by inhibitors of EGFR and MEK1/2. GIF-14 cells were treated with SB431542 (TGF-βRI inhibitor; 10 µM) or AG1478 (EGFR inhibitor; 10 µM) or U0126 (MEK1/2 inhibitor; 10 µM) or in combination with TGF-β1 (2.5 ng/ml) for 48 h. Changes in the mRNA levels of stemness marker <i>Hmga2</i> and EMT marker <i>Snai1</i> were ascertained by qRT-PCR and normalized values are expressed relative to the control values (means ± SEM, n = 4). Student's t-tests are performed where indicated. Single and double asterisks denote <i>p</i> value<0.05 and <i>p</i> value<0.01, respectively. (D) The effects of TGF-β1 and EGF on the phosphorylation states of Egfr and Erk. GIF-14 cells were treated with TGF-β1 (2.5 ng/ml; top panel) or murine EGF (10 ng/ml; bottom panel) for various short periods of time from 15 to 120 min. The expression levels of phosphorylated Egfr at tyrosine residues 1068 and 1092 and Erk1/2 were measured by Western blot analysis using pEgfr^Y1068/1092- and pErk1/2–specific antibodies Total Egfr expression was determined using anti-Egfr antibody #2 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070427#pone-0070427-g001" target="_blank">Figure 1F</a>. The expression level of α-tubulin serves as a control for the amount of proteins loaded.