human phopho ddr1 ddr2 y796 y740 antibody (R&D Systems)

R&D Systems human phopho ddr1 ddr2 y796 y740 antibody
Human Phopho Ddr1 Ddr2 Y796 Y740 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/phospho+ddr2+antibody/Human+Phospho-DDR1%2FDDR2+(DDR1+Y796%2C+DDR2+Y740)+Antibody/bio_rxiv__2024__08__15__607761-57-18-16
Average 93 stars, based on 1 article reviews

human phopho ddr1 ddr2 y796 y740 antibody - by Bioz Stars, 2026-10

93/100 stars

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rabbit monoclonal anti p ddr2 (R&D Systems)

R&D Systems rabbit monoclonal anti p ddr2

A ) Western blot analysis of DDR1 and <t>DDR2</t> 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.

Rabbit Monoclonal Anti P Ddr2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/phospho+ddr2+antibody/Human+Phospho-DDR1%2FDDR2+(DDR1+Y796%2C+DDR2+Y740)+Antibody/bio_rxiv__857904-69-23-27
Average 94 stars, based on 1 article reviews

rabbit monoclonal anti p ddr2 - by Bioz Stars, 2026-10

94/100 stars

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human phospho ddr2 y740 antibody (R&D Systems)

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The Human Phospho DDR2 Y740 Antibody from R D Systems is a rabbit monoclonal antibody to DDR2 This antibody reacts with human The Human Phospho DDR2 Y740 Antibody has been validated for the following applications

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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.

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

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.

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.

Techniques: Western Blot, Expressing, RNA Sequencing Assay

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.

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.

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

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.

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.

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

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.

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.

Techniques: Transfection, Staining, Western Blot, Expressing

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.

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.

Techniques: Western Blot, Expressing, Transfection

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.

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.

Techniques: Transfection, Western Blot, Expressing

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.

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.

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

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.

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.

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

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.

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.

Techniques:

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.

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.

Techniques: Western Blot, Expressing, Immunohistochemistry, Immunostaining

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.

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.

Techniques: Mutagenesis, Expressing

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