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

Journal: PLoS ONE

Article Title: CCN1 Secretion Induced by Cigarette Smoking Extracts Augments IL-8 Release from Bronchial Epithelial Cells

doi: 10.1371/journal.pone.0068199

Figure Lengend Snippet: (A) CSE (10%)-induced CCN1 secretion in the presence of the inhibitors for ER stress related signaling. Beas2B cells were treated with JNK inhibitor II (10 µM), SB203580 (10 µM) for P38 MAPK, Bay11-7082 (10 µM) for NF-κB, GSK PERK (1 µM) for PERK and γ-secretase inhibitor (10 µM). Next, cells were exposed to CSE (10%) and CCN1 secretion was determined using ELISA as the above. (B) Reduced CCN1 secretion after CSE in the p38 MAPK and JNK siRNA transfected Beas2B cells. Beas2B cells were transfected with siRNA first. After 24 h, these cells were exposed to CSE (10%). After another 24 h, CCN1 secretion was determined using ELISA. (C) CSE (10%) induced JNK and p38 MAPK phosphorylation in Beas2B cells, determined by Western Blot analysis. (D) Deletion of Ire-1α by siRNA reduced CSE triggered CCN1 secretion. Beas2B cells were transfected with Ire-1α siRNA, followed by CSE (10%) exposure. CCN1 secretion was determined using ELISA as the above. Inset: the efficiency of Ire-1α deletion using siRNA. (E) Beas2B cells were transfected with Ire-1α siRNA or control siRNA, followed by CSE (10%). After 8 h, cell lysate was subjected to Western Blot analysis. pJNK, total JNK, p-p38 MAPK and total p38 MAPK were analyzed. (F) Beas2B cells were treated with ER stress inducer, thapsigargin (1 µM). CCN1 release was analyzed using ELISA. To determine the signaling pathways involved in the ER stress inducer triggered CCN1 release, we pre-treated the cells using a variety of pathway inhibitors as shown, followed by thapsigargin (1 µM). (G) Similarly, the above samples from (B) were subjected to ELISA to determine CCN1 secretion in p38 MAPK and JNK siRNA treated Beas2B cells. (H) Blocking of Ire-1α and/or PERK suppressed CCN1 secretion induced by thapsigargin. Beas2B cells were transfected with control siRNA or Ire-1α siRNA, treated with solvent or PERK inhibitor (10 µM), followed by thapsigargin (1 µM). CCN1 secretion was measured as above mentioned. *p<.05 All figures above represented two or three independent experiments with similar results.

Article Snippet: CCN1 and β-actin antibody were purchased from Santa Cruz (Santa Cruz, CA) and JNK, p-38 MAPK, pJNK, p-p38 MAPK, Ire-1α, p-eIF2α, pLRP6, LPR6, Dvl2, Dvl3, β-catenin, axin, PDI, ERp44, ERp94 and BiP/GRP78 from Cell Signaling (Danvers, MA).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Blocking Assay