Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: Correlations between DDR2 expression and clinicopathologic features in HCC

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: The expression of DDR2 in cell lines and HCC tissues. a DDR2 mRNA expression in the immortalized normal human liver cell line L-02 and five hepatoma cell lines (MHCC-97H, HepG2, Huh-7, Hep3B and SMMC-7721) using qRT-PCR ( n = 3, * P < 0.01). b DDR2 protein expression in the immortalized normal human liver cell line L-02 and five hepatoma cell lines (MHCC-97H, HepG2, Huh-7, Hep3B and SMMC-7721) using immunoblotting ( n = 3, P < 0.01). c DDR2 mRNA expression in HCC tissues (T) and corresponding adjacent no tumorous tissues (N) using qRT-PCR ( n = 112, P < 0.01). d The protein expression of DDR2 and phosphorylated DDR2 (p-DDR2) in HCC tissues (T) and corresponding adjacent no tumorous tissues (N) using immunoblotting ( n = 112, P < 0.01, respectively)

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: Prognostic value of DDR2 protein expression in HCC and its correlation with phosphorylated DDR2 (p-DDR2) in HCC cells. a Overall survival for DDR2 protein expression (log-rank, P < 0.01). b Disease-free survival for DDR2 protein expression (log-rank, P < 0.01). c Immunoblotting analysis of DDR2 and p-DDR2 in Hep3B cells with DDR2 expressing plasmid transfection or control plasmid transfection. d Immunoblotting analysis of DDR2 and p-DDR2 in MHCC-97H cells with DDR2-shRNA transfection or control-shRNA transfection

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Control, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: Univariate prognostic analysis of overall survival and disease-free survival in HCC patients

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: Multivariate analysis of factors contributing to overall survival and disease-free survival in HCC patients

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: DDR regulates HCC cell mobility and EMT phenotype. a Cell migration and invasion as measured by Transwell assays were promoted by up-regulation of DDR2 in Hep3B cells as compared with control cells ( n = 6, P < 0.01). b Cell migration and invasion as measured by Transwell assays were inhibited by down-regulation of DDR2 in MHCC-97H cells as compared with control cells ( n = 6, P < 0.01). c Immunoblotting analysis of E-cadherin and Vimentin in Hep3B cells with DDR2 expressing plasmid transfection or control plasmid transfection ( n = 6, P < 0.01). d Immunoblotting analysis of E-cadherin and Vimentin in MHCC-97H cells with DDR2-shRNA transfection or control-shRNA transfection ( n = 6, P < 0.01)

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques: Migration, Control, Western Blot, Expressing, Plasmid Preparation, Transfection, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: The effect of DDR2 on the expression of SNAIL1, TWIST1 and ZEB1 in HCC cells. a Immunoblotting analysis of SNAIL1, TWIST1 and ZEB1 in Hep3B cells with DDR2 expressing plasmid transfection or control plasmid transfection ( n = 3, P < 0.01). b Immunoblotting analysis of SNAIL1, TWIST1 and ZEB1 in MHCC-97H cells with DDR2-shRNA transfection or control-shRNA transfection ( n = 3, P < 0.01). c qRT-PCR analysis of SNAIL1 in Hep3B cells with DDR2 expressing plasmid transfection or control plasmid transfection ( n = 3, * P < 0.01). d qRT-PCR analysis of SNAIL1 in MHCC-97H cells with DDR2-shRNA transfection or control-shRNA transfection ( n = 3, * P < 0.01). e Cycloheximide (CHX) treated samples analysis following DDR2 expressing plasmid transfection or control plasmid transfection in Hep3B cells ( n = 3, * P < 0.01). f Cycloheximide (CHX) treated samples analysis following DDR2-shRNA transfection or control-shRNA transfection in MHCC-97H cells ( n = 3, * P < 0.01)

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Control, shRNA, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: Immunohistochemical staining of DDR2 and SNAIL1 in HCC tissues. a The IHC scores of DDR2 in HCC tissues was notably higher than that in the corresponding adjacent no tumorous tissues (NT) ( n = 112, P < 0.01). b The IHC scores of SNAIL1 in HCC tissues was notably higher than that in the corresponding adjacent no tumorous tissues (NT) ( n = 112, P < 0.01). c In cases of high DDR2 protein expression, there was strong SNAIL1 protein expression in the same tissue section. Similarly, in the case of low DDR2 protein expression, there was no detectable SNAIL1 protein expression in the same tissue section (scale bar: 100 μm). d Linear regression analysis showed a significant positive correlation between ACK1 and p-ACK1 protein expression ( P < 0.01)

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

doi: 10.1186/s13046-015-0218-6

Figure Lengend Snippet: DDR2 induces EMT and up-regulates the expression of MTI-MMP and MMP2 through ERK2/SNAIL1 signaling in HCC cells. a Hep3B cells that had been transfected with DDR2 expressing plasmid or control plasmid, respectively, were subjected to western blotting for ERK1/2, p-ERK1/2, SNAIL1, Vimentin and E-cadherin. b MHCC-97H cells that had been transfected with control-shRNA, DDR2-shRNA, control-siRNA, ERK2-siRNA, control-siRNA or SNAIL1-siRNA, respectively, were subjected to western blotting for DDR2, ERK1/2, p-ERK1/2, SNAIL1, Vimentin and E-cadherin). c Co-immunoprecipitation assays showed that ERK2 was bound with SNAIL1 directly. d Hep3B cells that had been transfected with DDR2 expressing plasmid or control plasmid, respectively, were subjected to western blotting for ERK1/2, p-ERK1/2, SNAIL1, MTI-MMP and MMP2. e MHCC-97H cells that had been transfected with control-shRNA, DDR2-shRNA, control-siRNA, ERK2-siRNA, control-siRNA or SNAIL1-siRNA, respectively, were subjected to western blotting for DDR2, ERK1/2, p-ERK1/2, SNAIL1, MTI-MMP and MMP2. f HCC cells that had been transfected with indicated plasmids were added to plates coated with collagen I (2 mg/ml, BD Biosciences) for 8 h and western blotting were performed

Article Snippet: The following primary antibodies were used in the western immunoblotting assays: SNAIL1 (ab180714, Abcam, Cambridge, UK), Vimentin (ab137321, Abcam, Cambridge, UK), E-cadherin (ab15148, Abcam, Cambridge, UK), MT1-MMP (ab53712, Abcam, Cambridge, UK), DDR2 (sc-8989, Santa Cruz, CA, USA), p-DDR2 (MAB25382, R&D systems Inc, USA), ERK1/2 (sc-292838, Santa Cruz, CA, USA), p-ERK1/2 (sc-101760, Santa Cruz, CA, USA), MMP2 (sc-10736, Santa Cruz, CA, USA) and β-actin polyclonal antibody (sc-130656, Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, shRNA, Immunoprecipitation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Prevalence of Mutations in Discoidin Domain-Containing Receptor Tyrosine Kinase 2 (DDR2) in Squamous Cell Lung Cancers in Korean Patients

doi: 10.4143/crt.2016.347

Figure Lengend Snippet: Primer sequences and polymerase chain reaction conditions

Article Snippet: The blots were probed with an anti- DDR2 antibody (2538-DR, R&D Systems Inc., Minneapolis, MN), and anti–α-tubulin, –β-actin antibodies (sc-8035, Santa Cruz Biotechnology), and anti–phosphotyrosine-100, –Src and c-Src antibodies (Cell Signaling Technologies, Danvers, MA).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Prevalence of Mutations in Discoidin Domain-Containing Receptor Tyrosine Kinase 2 (DDR2) in Squamous Cell Lung Cancers in Korean Patients

doi: 10.4143/crt.2016.347

Figure Lengend Snippet: Radiological and histological features and discoidin domain-containing receptor tyrosine kinase 2 ( DDR2 ) mutation analysis of case 1. (A) Chest computed tomography (CT) in 2009 shows a 3 cm nonenhancing mass-like consolidation with a background of patchy fibrotic changes and scattered calcified and noncalcified sub-centimeter nodules in both lungs. (B) Chest CT in 2010 shows that the mass-like consolidation has increased in size to 6.2 cm. Numerous new cavitary and noncavitary nodules can be seen in both lungs. (C) Squamous cell carcinoma on hematoxylin and eosin section (×200). (D) Chromatogram of DDR2 shows a missense mutation, as depicted by the arrows, with a change in the amino acids from GTG (valine) to GAG (glutamic acid) on exon 15.

Article Snippet: The blots were probed with an anti- DDR2 antibody (2538-DR, R&D Systems Inc., Minneapolis, MN), and anti–α-tubulin, –β-actin antibodies (sc-8035, Santa Cruz Biotechnology), and anti–phosphotyrosine-100, –Src and c-Src antibodies (Cell Signaling Technologies, Danvers, MA).

Techniques: Mutagenesis, Computed Tomography

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Prevalence of Mutations in Discoidin Domain-Containing Receptor Tyrosine Kinase 2 (DDR2) in Squamous Cell Lung Cancers in Korean Patients

doi: 10.4143/crt.2016.347

Figure Lengend Snippet: Radiological and histological features and discoidin domain-containing receptor tyrosine kinase 2 ( DDR2 ) mutation analysis of case 2. (A) Chest computed tomography scan showing a 5.3-cm necrotic cavitary mass. (B, C) Squamous cell carcinoma on hematoxylin and eosin section (×40 and ×200, respectively). (D) p63 staining showing nuclear staining of tumor cells. (E) Thyroid transcription factor 1 staining showing negative staining of tumor cells. (F) Chromatogram of DDR2 showing a missense mutation, as depicted by the arrows, with a change in the amino acids from CTA (leucine) to CCA (proline) on exon 15.

Article Snippet: The blots were probed with an anti- DDR2 antibody (2538-DR, R&D Systems Inc., Minneapolis, MN), and anti–α-tubulin, –β-actin antibodies (sc-8035, Santa Cruz Biotechnology), and anti–phosphotyrosine-100, –Src and c-Src antibodies (Cell Signaling Technologies, Danvers, MA).

Techniques: Mutagenesis, Computed Tomography, Staining, Negative Staining

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Prevalence of Mutations in Discoidin Domain-Containing Receptor Tyrosine Kinase 2 (DDR2) in Squamous Cell Lung Cancers in Korean Patients

doi: 10.4143/crt.2016.347

Figure Lengend Snippet: Functional effects of discoidin domain-containing receptor tyrosine kinase 2 ( DDR2 ) mutants observed through in vitro assays. (A) Schematic view shows a known mutation and novel mutations (red circles) within the kinase domain of DDR2 . (B) Activation of DDR2 and Src molecules by the ectopic expression of DDR2 point mutants. NIH3T3 cells were stably expressed with the indicated DDR2 mutant constructs, stimulated with 10 μg/mL collagen, and then analyzed using western blot. WT, wild type. (C) In vitro transforming assay in soft agar. NIH3T3 cells expressing the indicated mutant protein were seeded in 6-well plates and cultured for 14 days in soft agar. The number of colonies formed per well, indicated as the mean±standard deviation, from the three wells in one representative experiment out of three. ***p < 0.001, scale bars=10 μm. (D) The effect of dasatinib in cells expressing DDR2 mutants. Dasatinib caused reduced proliferation and a decrease in phosphorylation of Src in cells expressing DDR2 V582E and L595P. In the left panel, the NIH3T3 cells expressing the DDR2 mutants were treated with the indicated doses of dasatinib for 72 hours, and cell viability was determined. ***p < 0.001. In the right panel, cells were treated with 0.1 μM dasatinib for 24 hours, followed by cell lysis and detection of the indicated protein using western blot. (E) In vitro colony-forming ability of cells expressing DDR2 mutants following dasatinib treatment. NIH3T3 cells expressing the DDR2 mutants were seeded in soft agar and cultured with or without dasatinib for 7 days. The colonies formed were stained with 0.001% crystal violet and the number of colonies was estimated. The images were obtained using a phase-contrast microscope at 40× magnification. The values shown represent the average of three independent experiments and error bars indicate standard deviations. *p < 0.05, ***p < 0.001.

Article Snippet: The blots were probed with an anti- DDR2 antibody (2538-DR, R&D Systems Inc., Minneapolis, MN), and anti–α-tubulin, –β-actin antibodies (sc-8035, Santa Cruz Biotechnology), and anti–phosphotyrosine-100, –Src and c-Src antibodies (Cell Signaling Technologies, Danvers, MA).

Techniques: Functional Assay, In Vitro, Mutagenesis, Activation Assay, Expressing, Stable Transfection, Construct, Western Blot, Cell Culture, Standard Deviation, Phospho-proteomics, Lysis, Staining, Microscopy

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Prevalence of Mutations in Discoidin Domain-Containing Receptor Tyrosine Kinase 2 (DDR2) in Squamous Cell Lung Cancers in Korean Patients

doi: 10.4143/crt.2016.347

Figure Lengend Snippet: Cancer-associated DDR2 mutations and responses to dasatinib

Article Snippet: The blots were probed with an anti- DDR2 antibody (2538-DR, R&D Systems Inc., Minneapolis, MN), and anti–α-tubulin, –β-actin antibodies (sc-8035, Santa Cruz Biotechnology), and anti–phosphotyrosine-100, –Src and c-Src antibodies (Cell Signaling Technologies, Danvers, MA).

Techniques: Mutagenesis