Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: TGF-β1 enhances HMGA1 expression in SW579 cells. (A) Real-time PCR analysis of HMGA1 expression induced in SW579 cells by treatment with various concentrations of TGF-β1 for 8 h. (B) Real-time PCR analysis of HMGA1 expression induced by treatment with 5 ng/ml of TGF-β1 for the specified time. (C–E) Immunofluorescence staining of HMGA1 expression induced by treatment with different concentrations of TGF-β1 for 8 h or with 5 ng/ml of TGF-β1 for the specified time in SW579 cells. Fluorescence were gathered and analyzed with a fluorescence microscope (Olympus). * P<0.05, compared to the group not treated with TGF-β1. Scale bar, 50 µ m.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: TGF-β1 induces HMGA1 expression through PI3K signaling and ERK signaling in SW579 cells. (A) The effects of wortmannin, PD98059 and U0126 on the mRNA expression of HMGA1 induced by treatment with 5 ng/ml of TGF-β1 in SW579 cells. (B and C) The effects of wortmannin, PD98059 and U0126 on the expression of HMGA1 induced by treatment 5 ng/ml with TGF-β1 in SW579 cells. Fluorescence were gathered and analyzed with a fluorescence microscope (Olympus). * P<0.05, compared to the group treated with TGF-β1. Scale bar, 50 µ m.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Expressing, Fluorescence, Microscopy

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: TGF-β1 enhances the promoter activity of HMGA1 in SW579 cells. (A) The effects of TGF-β1 on HMGA1 promoter activity. HMGA1 promoter, control vectors or pGL4.1 vectors were transfected into SW579 cells, and treated with the indicated concentrations of TGF-β1. (B) The effects of 5 ng/ml TGF-β1 on HMGA1 promoter activity. HMGA1 promoter, control vectors or pGL4.1 vectors were transfected into SW579 cells, and treated with 5 ng/ml TGF-β1 for the indicated time. (C) The effects of 5 ng/ml TGF-β1 on HMGA1 promoter activity with wortmannin (100 nM), PD98059 (20 µ M) or U0126 (25 µ M) in SW579 cells, * P<0.05. Luciferase activity was normalized by using a Renilla luciferase internal control. Each experiment was repeated in triplicate and performed at least twice.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Activity Assay, Transfection, Luciferase

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: Identification of the lentiviral vector with the highest knockdown efficiency. (A) Fluorescence microscopy was used to observe the infection efficiency of different lentiviral vectors in TPC1 cells (magnification, x100). a, TPC1 cells without lentiviral infection in the optical microscope (Con group); b, TPC1 cells of Con group in the fluorescence microscope; c, TPC1 cells were infected with negative lentivirus NC/GV248 RNAi-LV (NC group) in the optical microscope; d, TPC1 cells of NC group in the fluorescence microscope; e, TPC1 cells were infected with HMGA1/GV248RNAi-LV#3 RNAi (KD group) at a high MOI in the optical microscope; f, TPC1 cells of KD group at a high MOI in the fluorescence microscope. Scale bar, 50 µ m. (B) Fluorescence microscopy examination of the infection efficiencies of different lentiviral vectors in SW579 cells (magnification, x100). The panels are described as similar with that in (A). Scale bar, 50 µ m. (C) Relative levels of HMGA1 in SW579 cells infected with different groups of lentiviral particles. Either low or high MOI, HMGA1 expression significantly decreased in SW579 cells infected with different groups of lentiviral particles. The highest knockdown efficiency was obtained using KD3 lentiviral particles. * P<0.05, ** P<0.01.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Plasmid Preparation, Fluorescence, Microscopy, Infection, Expressing

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: Lentivirus-mediated HMGA1 knockdown inhibited TPC1 cell growth. (A) MTT assay showed that lentivirus-mediated HMGA1 knockdown significantly inhibited thyroid cancer TPC1 cell proliferation (n=3). ** P<0.01. (B) Lentivirus-mediated HMGA1 knockdown significantly inhibited thyroid cancer TPC1 cell colony formation (n=3, ** P<0.01). (C) The cells in the G 0 –G 1 and S phases significantly increased and the cells in the S phase significantly decreased in lentivirus-mediated HMGA1 knockdown group ( ** P<0.01; *** P<0.001).

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: MTT Assay

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: Lentivirus-mediated HMGA1 knockdown inhibited SW579 cell invasion and migration. (A) Lentivirus-mediated HMGA1 knockdown significantly inhibited thyroid cancer SW579 cell invasion. *** P<0.001. (B) Lentivirus-mediated HMGA1 knockdown significantly inhibited thyroid cancer SW579 cell migration, ** P<0.01. (C) Western blotting assay showed that lentivirus-mediated HMGA1 knockdown decreased MMP-2 expression, and increased E-cadherin expression in SW579 cells.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Migration, Western Blot, Expressing

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: Correlation of HMGA1 and MMP-2 expression with clinicopathological parameters in thyroid cancer.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Expressing, Significance Assay

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: HMGA1 protein expression correlates with MMP-2 expression in thyroid carcinoma (images in original magnification, x400). (A) Thyroid normal tissue. (B) Thyroid papillary carcinoma. (C) Thyroid follicular carcinoma. (D) Thyroid undifferentiated carcinoma.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: Expressions of HMGA1 and MMP-2 in thyroid normal tissue and cancer.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques:

Journal: International Journal of Oncology

Article Title: TGF-β1 induces HMGA1 expression: The role of HMGA1 in thyroid cancer proliferation and invasion

doi: 10.3892/ijo.2017.3958

Figure Lengend Snippet: Correlation of HMGA1 and MMP-2 expression in thyroid cancer.

Article Snippet: The rabbit polyclonal HMGA1 antibody (1:150; ab4078, Abcam) and MMP-2 antibody (1:100; ab37150, Abcam) were used.

Techniques: Expressing

Journal: International journal of cancer

Article Title: Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

doi: 10.1002/ijc.24675

Figure Lengend Snippet: FIGURE 2 – Increased Id-1 expression in KYSE150 esophageal cancer cells promoted tumor growth through PI3K/AKT pathway in nude mice. (a, b) Growth curves and representative photographs of subcutaneous tumors formed by inoculation of KYSE150-CON or KYSE150-Id-1 cells in nude mice. The mice bearing KYSE150-Id-1 xenografts (n 5 8) received 25 mg/kg LY294002 (n 5 8) or DMSO (n 5 8) through intraperitoneal injection thrice per week. Bars, SD; *p < 0.05, compared with DMSO-treated KYSE150-CON-injected mice. (c) Ki-67, CD31 immunostaining and TUNEL staining of tumor xenografts from the 3 groups (left panel); tumor proliferation index, tumor MVD and apoptosis index (right panel). Results represent mean 6 SD of at least 6 fields from representative tumor sections. *p < 0.05, **p < 0.005, ***p < 0.001, compared with DMSO-treated mice injected with KYSE150-CON cells. (d) Western blot analysis showed increased expression of Id-1, p-AKT, p-GSK3b, Bcl-2 and Bcl-xL, as well as decreased cleaved caspase-3, in the KYSE150-Id-1 tumor xenografts, compared with the KYSE150-CON tumor xenografts. Treatment with LY294002 (LY) attenuated the effect of Id-1 on AKT activation and its downstream targets. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Article Snippet: ESCC cell lines KYSE30, KYSE70, KYSE150 and KYSE270,21 obtained from DSMZ, were maintained in RPMI Abbreviations: AKT, protein kinase B; EMT, epithelial–mesenchymal transition; ESCC, esophageal squamous cell carcinoma; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GSK3b, glycogen synthase kinase 3b; HLH, helix-loop-helix; HMGA1, high mobility group A1; hTERT, human telomerase reverse transcriptase; Id-1, inhibitor of differentiation or DNA binding; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MVD, microvessel density; NFjB, nuclear factor-kappa B; PH, pleckstrin-homology; PI3K, phosphatidylinositol-3-kinase; PX, Phox; TMA, tissues microarrays; SDS, sodium dodecyl sulfate; TUNEL, TdTmediated dUTP nick-end labeling; VEGF, vascular endothelial growth factor.

Techniques: Expressing, Injection, Immunostaining, TUNEL Assay, Staining, Western Blot, Activation Assay

Journal: International journal of cancer

Article Title: Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

doi: 10.1002/ijc.24675

Figure Lengend Snippet: FIGURE 3 – Effects of Id-1 modulation on expression of EMT markers in esophageal cancer cells. (a) Id-1-overexpressing ESCC cells (KYSE150-Id-1, T.Tn-Id-1) and vector control cells (KYSE150-CON, T.Tn-CON) were compared for the expression of E-cadherin, b-catenin and N-cadherin by Western blot. Actin was included as loading controls. The Id-1-overexpressing cells showed lower expression of E-cadherin and b-catenin, but higher N- cadherin, compared with vector control cells and with the LY294002-treated Id-1-overexpressing cells. (b) Immunofluores- cence showing weaker staining of E-cadherin in Id-1-overexpressing KYSE150 cells compared with the control cells. Treatment with LY294002 led to upregulation of E-cadherin expression in KYSE150 cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Article Snippet: ESCC cell lines KYSE30, KYSE70, KYSE150 and KYSE270,21 obtained from DSMZ, were maintained in RPMI Abbreviations: AKT, protein kinase B; EMT, epithelial–mesenchymal transition; ESCC, esophageal squamous cell carcinoma; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GSK3b, glycogen synthase kinase 3b; HLH, helix-loop-helix; HMGA1, high mobility group A1; hTERT, human telomerase reverse transcriptase; Id-1, inhibitor of differentiation or DNA binding; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MVD, microvessel density; NFjB, nuclear factor-kappa B; PH, pleckstrin-homology; PI3K, phosphatidylinositol-3-kinase; PX, Phox; TMA, tissues microarrays; SDS, sodium dodecyl sulfate; TUNEL, TdTmediated dUTP nick-end labeling; VEGF, vascular endothelial growth factor.

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Staining

Journal: International journal of cancer

Article Title: Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

doi: 10.1002/ijc.24675

Figure Lengend Snippet: FIGURE 4 – Effects of Id-1 and PI3K/AKT inhibition on esophageal cancer cell migration, invasion and MMP expressions. (a) The ability of Id-1-overexpressing ESCC cells and the vector control cells grown in a confluent monolayer to migrate into a linear wound created by a pipette tip was monitored for 24 hr. Note the enhanced migration ability in the 2 esophageal cancer cell lines overexpressing Id-1. (b) Cellular invasive- ness of the esophageal cancer cells was assayed using Matrigel-coated Boyden chamber. Stable upregulation of Id-1 expression resulted in sig- nificant enhancement of cellular invasiveness in both KYSE150 and T.Tn cells. The effects were almost completely abolished by the addition of LY294002 (15 lM) to the upper compartment and partially counteracted by anti-MMP-9 neutralizing antibody (2 lg/ml). Bars, SD; *p < 0.005 and **p < 0.001, compared with DMSO-treated control cells. (c, d) Western blot and gelatin zymography analyses of MMP-2 and MMP-9 in Id-1-overexpressing ESCC cells (KYSE150-Id-1, T.Tn-Id-1) and vector control cells (KYSE150-CON, T.Tn-CON). Note that ectopic Id-1 expression induced both protein expressions and enzymatic activities of MMP-2 and MMP-9, and this induction was attenuated in a dose- dependent manner by LY294002 in both cell lines.

Article Snippet: ESCC cell lines KYSE30, KYSE70, KYSE150 and KYSE270,21 obtained from DSMZ, were maintained in RPMI Abbreviations: AKT, protein kinase B; EMT, epithelial–mesenchymal transition; ESCC, esophageal squamous cell carcinoma; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GSK3b, glycogen synthase kinase 3b; HLH, helix-loop-helix; HMGA1, high mobility group A1; hTERT, human telomerase reverse transcriptase; Id-1, inhibitor of differentiation or DNA binding; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MVD, microvessel density; NFjB, nuclear factor-kappa B; PH, pleckstrin-homology; PI3K, phosphatidylinositol-3-kinase; PX, Phox; TMA, tissues microarrays; SDS, sodium dodecyl sulfate; TUNEL, TdTmediated dUTP nick-end labeling; VEGF, vascular endothelial growth factor.

Techniques: Inhibition, Migration, Plasmid Preparation, Control, Transferring, Expressing, Western Blot, Zymography

Journal: International journal of cancer

Article Title: Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

doi: 10.1002/ijc.24675

Figure Lengend Snippet: FIGURE 5 – Experimental metastasis assay showing that ectopic Id-1 expression in KYSE150 esophageal cancer cells promoted tumor metas- tasis in vivo through the PI3K/AKT pathway. (a) KYSE150-CON cells pretreated with DMSO, and KYSE150-Id-1 pretreated with DMSO or LY294002, were intravenously injected into nude mice through the tail vein (n 5 8 per group). Representative images of lungs harvested 6 weeks postinjection are shown. Note that only the DMSO-treated KYSE150-Id-1 cells formed large metastatic nodules (arrows) in the lungs. (b) Sur- face metastatic nodules in the lungs were counted, and the numbers presented separately for nodules below and above 1 mm in diameter. Bars, SD; *p < 0.001 compared with control group injected with DMSO-pretreated KYSE150-CON cells. (c) Representative hematoxylin and eosin stained lung sections from the 3 groups of mice showing micrometastatic tumor cells (arrows) in the lungs. A human-specific anti-cytokeratin 8 (CK 8) monoclonal antibody was used to distinguish human cancer cells from the surrounding mouse pulmonary tissue. (d) The lungs from the 3 groups were compared for the expression of human cytokeratin 8 by Western blot. Human esophageal cancer cells KYSE150 and lung tissue from normal untreated mice were included as positive and negative controls, respectively. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Article Snippet: ESCC cell lines KYSE30, KYSE70, KYSE150 and KYSE270,21 obtained from DSMZ, were maintained in RPMI Abbreviations: AKT, protein kinase B; EMT, epithelial–mesenchymal transition; ESCC, esophageal squamous cell carcinoma; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GSK3b, glycogen synthase kinase 3b; HLH, helix-loop-helix; HMGA1, high mobility group A1; hTERT, human telomerase reverse transcriptase; Id-1, inhibitor of differentiation or DNA binding; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MVD, microvessel density; NFjB, nuclear factor-kappa B; PH, pleckstrin-homology; PI3K, phosphatidylinositol-3-kinase; PX, Phox; TMA, tissues microarrays; SDS, sodium dodecyl sulfate; TUNEL, TdTmediated dUTP nick-end labeling; VEGF, vascular endothelial growth factor.

Techniques: Expressing, In Vivo, Injection, Control, Staining, Western Blot