Twist is required for TrkB-induced EMT-like transformation and anoikis suppression.

<p>A: Quantification was performed to determine the relative change in Twist mRNA expression following transfection of Ishikawa^TrkB cells with control (siRNA-NT) or siRNA-Twist oligonucleotide duplexes. SiRNA-Twist#1 was chosen for further study (**<i>p</i><0.01). B. Invasion ability was significantly decreased in Ishikawa^TrkB cells transfected with siRNA-Twist compared to untreated cells or cells transfected with siRNA-NT as assessed by transwell assay(**<i>p</i><0.01; NS, not significant), Bars show mean ± SD. (Below, 200×). C. Protein levels of Twist, TrkB and EMT markers as analyzed by immunoblotting in Ishikawa^TrkB cells (Left), and further quantified by densitometry of triplicate blots (Right, **<i>p</i><0.01; #, not significant). β-actin was included as an internal control. E. Surviving Ishikawa^TrkB cell spheroids in suspension culture (Left, upper: 40×, below: 400×). After adhering for 4 h on glass coverslips, spheres were immunostained with antibodies against Twist (red) and TrkB (green). In the merged images (bottom right), these spheres contain double-positive cells (400×). Quantification of these cell aggregates formed by surviving Ishikawa^TrkB cells showed a higher number of TrkB⁺/Twist⁺ cells than non-expressing cells (Right panel). The data are reported as mean ± SD (**<i>p</i><0.01). All experiments were carried out in triplicate and repeated at least three times.