Fig 2: Twist is required for TrkB-induced EMT-like transformation and anoikis suppression.A: Quantification was performed to determine the relative change in Twist mRNA expression following transfection of IshikawaTrkB cells with control (siRNA-NT) or siRNA-Twist oligonucleotide duplexes. SiRNA-Twist#1 was chosen for further study (**p<0.01). B. Invasion ability was significantly decreased in IshikawaTrkB cells transfected with siRNA-Twist compared to untreated cells or cells transfected with siRNA-NT as assessed by transwell assay(**p<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 IshikawaTrkB cells (Left), and further quantified by densitometry of triplicate blots (Right, **p<0.01; #, not significant). ß-actin was included as an internal control. E. Surviving IshikawaTrkB 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 IshikawaTrkB cells showed a higher number of TrkB+/Twist+ cells than non-expressing cells (Right panel). The data are reported as mean ± SD (**p<0.01). All experiments were carried out in triplicate and repeated at least three times.

Fig 3: TrkB effects anoikis resistance in suspension culture.A. Representative images of flow cytometry analysis of apoptotic cells by Annexin V-PE/7-AAD staining in HEC-1B cell lines 24 h and 48 h after anchorage-independent culture. B. Representative images of flow cytometry analysis of apoptotic cells by Annexin V-PE/7-AAD staining in Ishikawa cell lines 24 h and 48 h after anchorage-independent culture. C and D. Flow cytometry results were plotted as the mean+SD of triplicate experiments at 24 h and 48 h after suspension culture. **p<0.01 considered significant; NS, not significant. All experiments were carried out in triplicate and repeated at least three times.

Fig 4: Inhibition of TrkB in the dorsal striatum blocked the therapeutic effects of 7,8-DHF. (A) Experimental designs. In Exp.#1, CON-siRNA or TrkB-siRNA was injected in the dorsal striatum (AP, +1.0; ML, ±1.5; DV, -3.6 mm) of normally raised WT mice, and the mice were sacrificed on day 3. In Exp.#2, CON-siRNA or TrkB-siRNA was injected in the dorsal striatum of normally raised WT mice or ELS-exposed D2+/- mice, followed by 7,8-DHF injection (i.p., 10 mg/kg/day) and subsequent behavioral tests. (B, C) Western blots showing expression levels of TrkB, p-AKT, AKT, p-ERK1/2, ERK1/2, and ß-Actin in the dorsal striatum injected with TrkB-siRNA or CON-siRNA (B). Samples were collected 2 days after siRNA injection. Quantification of Western blots (C). n= 4 animals, each, 3–4 repeats (TrkB: p=0.0002; p-AKT/AKT: p=0.0367; p-ERK/ERK: p=0.0014). (D~F) The amount of time spent in each chamber in the three-chamber test (D), social interaction time in the home-cage social interaction test (E), and grooming time (F) of normally raised WT mice and ELS-exposed D2+/- mice injected with TrkB-siRNA or CON-siRNA in the dorsal striatum and treated with vehicle or 7,8-DHF as indicated. n=5~6 animals (three-chamber test: WT, CON+siCON+Veh: p=0.0017; WT, CON+siTrkB+Veh; p=0.0015; D2+/-, ELS+siCON+Veh: p=0.5463, D2+/-, ELS+siCON+DHF: p=0.0025, D2+/-, ELS+siTrkB+DHF: p=0.9304; Grooming, WT, CON+siCON+Veh vs. WT, CON+siTrkB+Veh: p=0.9081; D2+/-, F(2, 14)=0.2332, p=0.7950). Data are presented as mean±SEM. * and ** denote differences between the indicated groups at p<0.05 and p<0.01, respectively (Student's t-test or one-way ANOVA and Holm-Sidak post hoc test).

Fig 5: TrkB induce EMT in EC cell lines.A. Left: Mesenchymal morphology induced by over-expression of TrkB in Ishikawa cells (200×). Right: Epithelial morphology induced by suppression of TrkB in HEC-1B cells (200×). B. Messenger RNA (mRNA) levels of TrkB and EMT markers as analyzed by qRT-PCR in Ishikawa cells. ß-actin was included as an internal control. (*p<0.05, **p<0.01; #, not significant). C. Protein levels of TrkB and EMT markers in Ishikawa cells as analyzed by immunoblotting (Right), and further quantified by densitometry of triplicate experiments (Left, **p<0.01; #, not significant). ß-actin was included as an internal control. D. mRNA levels of TrkB and EMT markers as analyzed by qRT-PCR in HEC-1B cells (*p<0.05, **p<0.01; #, not significant). E. Protein levels of TrkB and EMT markers in HEC-1B cells as analyzed by immunoblotting (Right), and further quantified by densitometry of triplicate experiments (Left, **p<0.01; #, not significant). ß-actin was included as an internal control. All experiments were carried out in triplicate and repeated at least three times.