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: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) Western blot analysis of DDR1 and DDR2 expression in 3 benign nevi (N1, N2, N3), 15 metastatic melanoma patient biopsies (1–15), and in 14 melanoma cell lines. β-actin was used as the endogenous loading control. B ) DDR expression in melanoma progression from a metaanalysis of 363 cutaneous melanomas from TCGA database analysis (skin cutaneous melanoma, PanCancer Atlas. C ) RNA sequencing data ( GSE50535, GSE5050 ) for DDR1 or DDR2 mRNA expression before and after vemurafenib treatment. D ) Western blot analysis of DDR1 and DDR2 expression in a subset of three melanoma cell lines (229, 238, 249) either sensitive (S) or resistant (R) to vemurafenib. GAPDH was used as the endogenous loading control. The graph shows the quantification of DDR expression. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05, ns: non-significant. E ) Western blot analysis of DDR1 and DDR2 expression in 229 S cells treated with vemurafenib (10 nM), cobimetinib (10 nM), or both over 2 months. GAPDH was used as the endogenous loading control.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) Western blot analysis of DDR1 and DDR2 expression in 15 metastatic melanoma patient biopsies. β-actin was used as the endogenous loading control. B) Summary table of the different cell lines used in the screening experiment as well as the mutations found in these cell lines. C ) RNA sequencing data ( GSE50509 ) for DDR1 or DDR2 mRNA expression before and after treatment with an anti-BRAF.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) RNA sequencing data ( GSE65185 ) for AXL, DDR1 and DDR2 mRNA expression in vemurafenib sensitive or resistant cell lines. B ) Western blot analysis of AXL, MITF, DDR1, and DDR2 expression in a subset of three melanoma cell lines (229, 238, 249) sensitive (S) or resistant (R) to vemurafenib. GAPDH was used as the endogenous loading control. The graph shows the quantification of AXL and MITF expression. Values are expressed as the mean ± SEM of three independent experiments. ns: non-significant, *p<0.05. C ) 238 R cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), or 238 R cells were treated with DDR1 inhibitor. Protein extracts were then analyzed by immunoblotting to determine PDDR1, DDR1, and AXL expression. The graph shows the quantification of AXL/GAPDH expression. GAPDH was used as the endogenous loading control. Values are expressed as the mean ± SEM of three independent experiments. ns: non-significant. D ) 238 R cells were transfected with an siRNA control (siGl2) or targeting DDR2 (siDDR2), or 238 R cells were treated with DDR2 inhibitor. Protein extracts were then analyzed by immunoblotting to determine PDDR2, DDR2, and AXL expression. GAPDH was used as the endogenous loading control. The graph shows the quantification of AXL/GAPDH expression. Values are expressed as the mean ± SEM of three independent experiments. **p<0.01, ***p<0.001. E ) 238 S cells were transiently transfected with DDR2-mCherry. Protein extracts were then analyzed by immunoblotting to determine DDR2 and AXL expression. GAPDH was used as the endogenous loading control. The graph shows the quantification of DDR expression. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. F ) 238 R cells were transfected with an siRNA control (siGl2) or targeting AXL (siAXL1/2). Protein extracts were then analyzed by immunoblotting to determine AXL, DDR2, and MITF expression. GAPDH was used as the endogenous loading control. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05, ns: non-significant.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: RNA Sequencing Assay, Expressing, Western Blot, Transfection

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) RNA sequencing data (GSE4845, GSE4843, GSE4840) for DDR1 and DDR2 mRNA expression in proliferative or invasive melanoma cells. B ) 229 R cells were treated with DDR2 inhibitor. Protein extracts were then analyzed by immunoblotting to determine PDDR2, DDR2, and AXL expression. GAPDH was used as the endogenous loading control. The graph shows the quantification of AXL/GAPDH expression. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. C ) Study of the interaction between AXL and DDR2 by co-immunoprecipitation (IP) assay in 238 R cells. AXL or DDR2 were immunoprecipitated using AXL or mCherry antibodies. D ) Other pathways commonly enriched in the following conditions: 238 R cells transfected with siRNA targeting DDR2 or treated with DDR2 inhibitor. Gene set enrichment analysis (GSEA) was performed against the Ingenuity Pathways database (Fisher’s Exact test expressed in −log10pvalue). E ) 249 R cells were transiently transfected with DDR2-mCherry. Representative images of the actin cytoskeletons of 249 R DDR2 mCh cells. Fluorescent staining corresponds to F-actin (green) and nuclei (blue). Scale bar: 6.78 uM. Protein extracts were then analyzed by immunoblotting to determine DDR2 expression. GAPDH was used as the endogenous loading control. F ) 238 S cells were transiently transfected with DDR2-mCherry. 238 (S, S DDR2-mCh, R) cells were seeded in Matrigel-coated chambers and invasion was assessed. The graph shows invasion index quantification. Values are expressed as the mean ± SEM of three independent experiments. *p = 0.0221; **p = 0.0057.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: RNA Sequencing Assay, Expressing, Western Blot, Immunoprecipitation, Transfection, Staining

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A) Bubble plot of the pathways commonly and significantly enriched between these conditions: 238 R cells transfected with siRNA targeting DDR2 or treated with DDR2 inhibitor. The bubble plot represents the ratio of control/siDDR2 or control/DDR2 inhibitor. Gene set enrichment analysis (GSEA) was performed against the Ingenuity Pathways database (Fisher’s Exact test expressed in −log10pvalue). Colors and dot size represent minus logarithms of adjusted p-values (padj). Column height represents the numbers of genes enriched in a pathway. B ) Representative images of parental versus resistant cell actin cytoskeletons. Fluorescent staining corresponds to F-actin (red) and nuclei (blue). Scale bar: 6.78 μM. C ) Representative images of the actin cytoskeleton of resistant invasive cells transfected with siRNA targeting DDR2 or treated with DDR2 inhibitor. Fluorescent staining corresponds to F-actin (red) and nuclei (blue). Scale bar: 6.78 μM. F-actin and G-actin in each condition were separated and measured using immunoblot analysis. The graph shows the quantification of F-actin/G-actin ratio. Values are expressed as the mean ± SEM of three independent experiments, *p<0.05, **p<0.01. D ) 238 R cells were transfected with an siRNA control (siGl2) or targeting DDR2 (siDDR2), or 238 R cells were treated with DDR2 inhibitor. Protein extracts were then analyzed by immunoblotting to determine PDDR2, DDR2, and PMLC2 expression. GAPDH was used as the endogenous loading control. E ) 238 S cells were transiently transfected with DDR2-mCherry. Representative images of the actin cytoskeletons of 238 S or 238 S DDR2 mCh cells. Fluorescent staining corresponds to F-actin (green) and nuclei (blue). Scale bar: 6.78 μM. Protein extracts were then analyzed by immunoblotting to determine DDR2 expression. GAPDH was used as the endogenous loading control. F ) 229 S cells or 249 S cells were transiently transfected with DDR2-mCherry. 229 (S, S DDR2-mCh, R) cells and 249 (S, R DDR2-mCh, R) were seeded in a Matrigel-coated chambers and invasion was assessed. The graph shows invasion index quantification. Values are expressed as the mean ± SEM from three independent experiments. *p = 0.0221; **p = 0.0057.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Transfection, Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) Western blot analysis of PErk and Erk expression in a subset of three melanoma cell lines (229, 238, and 249) either sensitive (S) or resistant (R) to vemurafenib. The graph shows the quantification of PErk/Erk expression. Values are expressed as the mean ± SEM of three independent experiments *p<0.05. B ) 238 R (left panel) cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), DDR2 (siDDR2), or both (siDDR1&2). Protein extracts were then analyzed by immunoblotting to determine PErk and Erk expression. GAPDH was used as the endogenous loading control. The graph shows the quantification of PErk/Erk expression. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05, **p<0.01. C ) mRNA expression level of MAP kinase targets in 238 R cells. The graph shows the quantification of PHLDA1, SPRY2, DUSP6, DUSP4, ETV4, and ETV5 mRNA expression level. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05, ***p<0.001, ns: non-significant. D ) 238 R cells were treated with DDR2 inhibitor (CR-13452) for 3 days. Protein extracts were then analyzed by immunoblotting to determine PErk, Erk, PDDR2, and DDR2 expression. GAPDH was used as the endogenous loading control. The graph shows the quantification of the ratio of PDDR2/DDR2 and PErk/Erk expression. **p<0.01. E ) 238 R cells were treated with dasatinib for 2 hours. Protein extracts were then analyzed by immunoblotting to determine PErk, Erk, DDR1, and DDDR2 expression. GAPDH was used as the endogenous loading control. The graph shows the quantification of the ratio of PDDR1/DDR1, PDDR2/DDR2, and PErk/Erk expression. Values are expressed as the mean ± SEM of three independent experiments. **p<0.01.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Transfection

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A) 229 R cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), DDR2 (siDDR2), or both (siDDR1&2). Protein extracts were then analyzed by immunoblotting to determine PErk and Erk expression. GAPDH was used as the endogenous loading control. B ) mRNA expression of MAP kinase targets in 229 R cells. The graph shows quantification of PHLDA1, SPRY2, DUSP6, DUSP4, ETV4, and ETV5 expression. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. C ) 238 R cells were treated with DDR1 inhibitor (7rh) for 3 days. Protein extracts were then analyzed by immunoblotting to determine PErk, Erk, PDDR1, and DDR1 expression. The graph shows the quantification of the ratio of PDDR1/DDR1 and PErk/Erk expression. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. D ) 229 R cells were treated with dasatinib for 2 hours. Protein extracts were then analyzed by immunoblotting to determine PErk and Erk expression. GAPDH was used as the endogenous loading control. The graph shows the quantification of the ratio of PDDR1/DDR1, PDDR2/DDR2, and PErk/Erk expression. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05, **p<0.01.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Transfection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) Left panel: Incucyte® proliferation assay analysis of 238 R cells seeded at 5 000 cells per well in a 96-well plate. The cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), DDR2 (siDDR2), or both (siDDR1&2). Values are expressed as the mean ± SEM of three independent experiments. **p<0.01. Right panel: Incucyte® apoptosis assay analysis of 238 R cells seeded at 5 000 cells per well in a 96 well plate. The cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), DDR2 (siDDR2), or both (siDDR1&2). Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. B ) Incucyte® proliferation assay analysis of 249 R cells seeded at 5 000 cells per well in a 96-well plate. The cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), DDR2 (siDDR2), or both (siDDR1&2). Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. C ) Left panel: Incucyte® proliferation assay analysis of 229 R cells seeded at 5 000 cells per well in a 96-well plate cultured in the presence or absence of DDR2 inhibitor. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. Right panel: Incucyte® apoptosis assay analysis of 229 R cells seeded at 5 000 cells per well in a 96-well plate cultured in the presence or absence of DDR2 inhibitor. Values are expressed as the mean ± SEM of three independent experiments. **p<0.01. D ) Left panel: Incucyte® proliferation assay analysis of 238 R cells seeded at 5 000 cells per well in a 96-well plate cultured in the presence or absence of 100 nM dasatinib. Values are expressed as the mean ± SEM of three independent experiments. **p<0.01. Right panel: Incucyte® apoptosis assay analysis of 238 R cells seeded at 5 000 cells per well in a 96 well plate cultured in the presence or absence of dasatinib (100 nM). Values are expressed as the mean ± SEM of three independent experiments. *p<0.05.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Proliferation Assay, Transfection, Apoptosis Assay, Cell Culture

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) Left panel: Incucyte ® proliferation assay analysis of 229 R cells seeded at 5 000 cells per well in a 96-well plate. The cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), DDR2 (siDDR2), or both (siDDR1&2). *p<0.05. Right panel: Incucyte ® apoptosis assay of 229 R cells seeded at 5 000 cells per well in a 96-well plate. The cells were transfected with an siRNA control (siGl2) or targeting DDR1 (siDDR1), DDR2 (siDDR2), or both (siDDR1&2). Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. B ) Left panel: Incucyte® proliferation assay analysis of 238 R cells seeded at 5 000 cells per well in a 96-well plate and cultured in the presence or absence of DDR1 inhibitor. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. Right panel: Incucyte® apoptosis assay analysis of 238 R cells seeded at 5 000 cells per well in a 96-well plate and cultured in the presence or absence of DDR1 inhibitor. Values are expressed as the mean ± SEM of three independent experiments. **p<0.01. C ) Incucyte® proliferation assay analysis of 238 R cells seeded at 5 000 cells per well in a 96-well plate and cultured in the presence or absence of DDR2 inhibitor. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. D ) Left panel: Incucyte® proliferation assay analysis of 229 R cells seeded at 5 000 cells per well in a 96-well plate and cultured in the presence or absence of 100 nM dasatinib. Values are expressed as the mean ± SEM of three independent experiments. **p<0.01. Lower panel: Incucyte® apoptosis assay analysis of 229 R cells seeded at 5 000 cells per well in a 96-well plate and cultured in the presence or absence of dasatinib (100 nM). Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. E) Bubble plot of the pathways commonly and significantly enriched between the following conditions: 238 R cells transfected with siRNA targeting DDR2 or treated with dasatinib. The bubble plot represents the ratio of control/siDDR2 or control/dasatinib. Gene set enrichment analysis (GSEA) was performed against the Ingenuity Pathways database (Fisher’s Exact test expressed in −log10pvalue). Colors and dot size represent minus logarithms of adjusted p-value (padj). Column height represents numbers of genes enriched in a pathway.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Proliferation Assay, Transfection, Apoptosis Assay, Cell Culture

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A) 229R, 238R, and 249R cells were seeded to form spheroids. B ) 229 R cell spheroids were treated after 72 h with DDR1 inhibitor at 0.8 μM. The graph shows the quantification of spheroid area in the different conditions. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05. C ) 229 R cell spheroids were treated after 72 h with DDR2 inhibitor at 5 μM. The graph shows the quantification of spheroid area in the different conditions. Values are expressed as the mean ± SEM of three independent experiments. **p<0.01. D ) 238 R cells were seeded to form spheroids and treated after 72 h with dasatinib at 100 nM. The graph shows the quantification of spheroid area in the different conditions. Values are expressed as the mean ± SEM of three independent experiments. *p<0.05.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques:

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) 229 R cells (5*10 ) were subcutaneously implanted into the right flanks of anesthetized 8-week-old NOD/LtSz- scid IL2Rγ null (NSG) mice. Mice were treated with vemurafenib until the tumors reached approximately 150 mm in volume, then the mice were randomly assigned into 2 groups: one control group treated with ongoing treatment with vemurafenib (40 mg/kg), and a second group with mice treated with dasatinib (20 mg/kg) (n=5 in each group). B ) Tumor growth of 229 R cells in the right flanks of mice. C ) Photographs of mice treated with vemurafenib or dasatinib. D ) Western blot analysis of DDR1, DDR2, PDDR1, and PDDR2 expression in primary tumors treated with or without dasatinib. GAPDH was used as the endogenous loading control. E ) Graphic representation of mice presenting metastasis under vemurafenib or dasatinib treatment. F ) Immunohistochemistry of primary tumors treated with or without dasatinib. Left panel: HES of primary tumor. Right panel: Immunostaining of Annexin V.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Immunohistochemistry, Immunostaining

Journal: bioRxiv

Article Title: Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching

doi: 10.1101/857904

Figure Lengend Snippet: A ) Following BRAF/MEK inhibitor treatment, BRAF-mutant melanoma cell lines undergo phenotype switching and became invasive via DDR2 upregulation. This in turn could i) regulate AXL expression and ii) activate the RhoA signaling pathway promoting cytoskeletal modifications. Once this resistant invasive phenotype is acquired, melanoma cells require upregulation of DDR1 and DDR2 to promote proliferation, but only DDR2 is able to overactivate the MAP kinase pathway. B ) Proposed model for the treatment of patients with BRAF-mutant metastatic melanoma. For vemurafenib-resistant patients, we propose post-treatment biopsy analysis. If DDR expression is detected at similar levels as to before treatment, we put forward dasatinib as an alternative treatment.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-DDR1 (5583S, Cell Signaling), rabbit monoclonal anti-DDR2 (12133S, Cell Signaling), rabbit monoclonal anti-P-DDR1 (14531, Cell Signaling), rabbit monoclonal anti-P-DDR2 (MAB25382, R&D Systems), mouse monoclonal anti-P-Erk (9106S, Cell Signaling); rabbit monoclonal anti-Erk (9102S, Cell Signaling); rabbit monoclonal anti-P-AKt (2965S, Cell Signaling), rabbit monoclonal anti-Akt (9272S, Cell Signaling), anti-AXL (8661S, Cell Signaling), anti-MITF (sc-56725, Santa Cruz) and mouse monoclonal anti-GAPDH (sc-166545, Santa Cruz Biotechnology).

Techniques: Mutagenesis, Expressing