Journal: Yonsei medical journal

Article Title: Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1α.

doi: 10.3349/ymj.2015.56.6.1503

Figure Lengend Snippet: Fig. 4. Knockdown of HIF-1α in FTC133 cells reverses hypoxia-induced EMT. Stable silencing of HIF-1α was performed using shRNA lentiviral particles (shHIF-1α). The lentiviral vector was used as the empty control vector (LVcontrol). All cells were incubated under normoxia or hypoxia (1% O2) for 16 h. (A) shHIF-1α transduction repressed hypoxia-induced HIF-1α activation in FTC133 cells. HIF-1α and EMT marker (E-cadherin, vimentin, and Twist) ex- pression was evaluated by (B) Western blot analysis, (C) real-time PCR using GAPDH as an internal control, and (D) immunofluorescence analysis in HIF-1α knockdown FTC133 cells. DAPI was used to label cell nuclei (blue). Scale bar=20 μm. (E) Transwell invasion assay and (F) scratch-wound heal- ing assay revealed that HIF-1α suppression significantly decreased the invasion and migration of FTC133 cells. Each panel was representative of trip- licate experiments. Scale bars=250 μm and 200 μm, respectively. Graphs show quantification of each assay. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01. HIF-1α, hypoxia inducible factor-1α; EMT, epithelial-to-mesenchymal transition; FTC, follicular thyroid cancer; LVcontrol, lentiviral control; NS, not significant.

Article Snippet: Lentivirus-mediated short hairpin RNA (shRNA) silencing of HIF-1α: Stable FTC133 cells were generated by polybrene (Santa Cruz Biotechnology, Inc., CA, USA) transduction using HIF-1α shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., CA, USA).

Techniques: Knockdown, shRNA, Plasmid Preparation, Control, Incubation, Transduction, Activation Assay, Marker, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Transwell Invasion Assay, Migration

Journal: Yonsei medical journal

Article Title: Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1α.

doi: 10.3349/ymj.2015.56.6.1503

Figure Lengend Snippet: Fig. 5. Knockdown of HIF-1α by specific shRNA reduces EMT in an orthotopic FTC murine model. To determine whether the aforementioned results were evident in vivo, stable shHIF-1α FTC133 cells (1×105/mouse) were injected into the right side of the thyroid gland in 6-week-old male nude mice (n=5). Twenty-eight days after inoculation with FTC133 cells, tumors were harvested for Western blotting and histologic evaluations including H&E staining and immunohistochemical analysis. (A) Representative image of tumors. Tumors derived from shHIF-1α cells showed significantly decreased tumor volume and weight compared with that from LVcontrol cells. Scale bar=10 mm. (B) H&E stain of tumor derived LVcontrol cells showed more in- vasive features than that from shHIF-1α cells. Upper panel (×100): tumors derived from shHIF-1α cells had a clear boundary between the tumor and adjacent nontumor tissue (arrowheads), whereas tumors induced by LVcontrol cells demonstrated irregular invasive fronts. Lower panel (×200): tu- mor cell invasions into blood vessel forming tumor embolis (arrows) are noted in the tissue from LVcontrol group, whereas no definite tumor emboli in that from shHIF-1α group. Scale bar=50 μm. (C) Representative immunohistochemical results showed more intense staining of E-cadherin, but weak- er staining of vimentin and Twist, supporting the in vitro results. Scale bar=100 μm. (D) Western blot analysis of HIF-1α, E-cadherin, vimentin, and Twist showed similar changes to those seen in vitro. **p<0.01, ***p<0.001. M, muscle; Tu, tumor; V, vessel; HIF-1α, hypoxia inducible factor-1α; EMT, epithe- lial-to-mesenchymal transition; FTC, follicular thyroid cancer; LVcontrol, lentiviral control; H&E, hematoxylin and eosin.

Article Snippet: Lentivirus-mediated short hairpin RNA (shRNA) silencing of HIF-1α: Stable FTC133 cells were generated by polybrene (Santa Cruz Biotechnology, Inc., CA, USA) transduction using HIF-1α shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., CA, USA).

Techniques: Knockdown, shRNA, In Vivo, Injection, Western Blot, Staining, Immunohistochemical staining, Derivative Assay, In Vitro, Control