Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 1. (a) Schematic diagram showing the structure of full-length DDR2, human DDR2-Fc, and mouse DDR2-V5-His constructs. SS, signal sequence; DS, discoidin domain; ECD, extracellular domain; IJXM, intracellular juxtamembrane region; TMD, transmembrane domain; ICD, intracellular domain; KD, kinase domain. (b) Purified recombinant DDR2-V5-His and DDR2- Fc proteins (20 ng/lane) were resolved by SDS-PAGE under reducing conditions (+βME), either with 4%–12% (w/v) Bis–Tris gels (left panel) or under reducing and non-reducing (−βME, 100 ng/lane) conditions with 10% SDS-PAGE (right panel). The separated protein was detected by immunoblotting using anti-epitope or anti-DDR2 antibodies as indicated.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Construct, Sequencing, Purification, Recombinant, SDS Page, Western Blot

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 2. (a) Solid-phase binding of DDR2 ECD proteins to immobilized bovine-dermal collagen I as indicated. Binding was detected using antibodies against DDR2 ECD. (b) Inhibition of fibrillogenesis of bovine-dermal collagen I assessed using turbidity measurements. DDR2 ECD proteins (40 μg/ml) as indicated were incu- bated with 200 μg/ml of neutralized collagen I in 96-well plates at 37 °C.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Binding Assay, Inhibition

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 3. AFM height images of monomeric DDR2-V5-His and dimeric DDR2-Fc before and after binding to bovine-dermal collagen I as indicated (a–d). DDR2-V5-His and DDR2-Fc particles bound to collagen are indicated by black and white arrows, respectively. Particle size distribution and average sizes are indicated in the accompanying histograms in panels e and f and in Table 1.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Binding Assay

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 4. Live cell imaging of DDR1b-YFP- (a–d) and DDR2-GFP- (e–g) expressing MC3T3-E1 cells using wide-field fluorescence microscopy, before and after the addition of collagen “C” as indicated. Insets (in panels a–g) show selected regions, which have been magnified from the corresponding images to visualize receptor assemblies. The location of these selected regions on the cell surface is indicated by dashed boxes. Endogenous DDR expression in MC3T3-E1 cells was evaluated using Western blotting panel h. Lysates of MC3T3-E1 cells were resolved by reducing 7.5% SDS-PAGE followed by immunoblot analyses using the indicated DDR antibodies. Asterisk (*) shows a non-specific band. Fluorescence microscopy images (a–c) show that DDR1b-YFP exhibits a uniform distribution on the cell surface before collagen stimulation and results in cluster formation upon collagen stimulation. Quantitative analysis (i) indicates that the number of punctuate structures in cells significantly increases upon collagen stimulation (*p b 0.05) and persist at 4 h. After 4 h of collagen stimulation, a subpopulation of DDR1b-YFP-expressing cells also exhibits the presence of long, filamentous structures (d). DDR2-GFP exhibits a uniform distribution on the cell surface before collagen stimulation (e) and does not result in cluster formation upon collagen stimulation (f). However, at 4 h post-collagen administration, filamentous structures were also observed in DDR2-GFP-expressing cells (g). The distribution of uninterrupted contour length of filamentous structures formed in DDR1b-YFP- and DDR2-GFP-expressing cells after 4 h of collagen stimulation is shown in panel j.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Live Cell Imaging, Expressing, Fluorescence, Microscopy, Western Blot, SDS Page

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 6. ICC performed on non-permeabilized cells for evaluating the association of collagen I with filamentous structures formed in (a) DDR1b-YFP- and (b) DDR2-GFP-expressing cells after 4 h of collagen stimulation. Total receptor is indicated in YFP channel (green), while staining with collagen antibodies is shown by TRITC (red). Co-localization YFP and TRITC is shown in yellow. Blue represents nuclear (DAPI) staining. Insets (in first row of each panel) show selected regions, which have been magnified from the corresponding images. Selected regions from three different cells are shown in the second row in each panel. YFP/GFP-positive filamentous structures had a very similar morphology as collagen fibrils and were observed to anchor the fibrils at cell edges. Collagen staining was intermittently present on or interspersed with YFP/GFP- positive filamentous structures.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Expressing, Staining

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 7. Western blotting of DDR expression and phosphorylation in MC3T3 cells. MC3T3-E1 cells were transiently transfected with (a) DDR1b-YFP or (b) DDR2-GFP expression vectors and stimulated with 20 μg/ml collagen I (+), as described in Materials and Methods. After 4 h of collagen stimulation, the cells were lysed in RIPA buffer and equal protein concentrations (25 μg/lane) were resolved by reducing 7.5% SDS-PAGE followed by immunoblot analyses using the indicated antibodies to DDR1 or DDR2. β-Actin was used as loading control. Number 1 in panel a indicates an additional band detected with D1G6 antibodies (discussed in the Results section). Asterisks (*) show non-specific bands.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Western Blot, Expressing, Phospho-proteomics, Transfection, SDS Page, Control

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 10. ICC performed on permeabilized cells for evaluating spatial distribution of receptor phosphorylation in DDR2- GFP-expressing cells after (a) 30 min and (b) 4 h of collagen stimulation. Total receptor is indicated in GFP channel (green), while staining with pDDR2 (Y740) antibodies is shown by TRITC (red). Co-localization GFP and TRITC is shown in yellow. Blue represents nuclear (DAPI) staining. Insets (in top row in each panel) show selected regions, which have been magnified from corresponding images. Little to no co-localization signal was detected, 30 min after collagen stimulation (a). At prolonged collagen stimulation (4 h), a number of filamentous structures formed in DDR2-GFP expressing cells co-localized with Y740 signal (b). The second row in panel b consists of selected regions from three different cells showing co-localization of filamentous structures with Y740 signal. A weak signal for Y740 (arrows) without a corresponding GFP signal can be observed in insets in (b, top row), which could correspond to endogenous pDDR2.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Phospho-proteomics, Expressing, Staining

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Scheme 1. Postulated model for spatial distribution and phosphorylation of DDRs upon collagen stimulation. DDR1 and DDR2 exist as homodimers on the cell surface. (a) Upon addition of monomeric collagen I, DDR1 and DDR2 interact with collagen, which leads (arrow 1) to the assembly of the receptors into filamentous structures aligned with collagen fibrils. This process occurs at prolonged (~4 h) times after collagen I stimulation. Phosphorylation of both DDR1 and DDR2 (arrow 2) co- localizes in these structures, as determined using Y513 (pDDR1b) as well as Y792 (pDDR1) and Y740 (pDDR2) antibodies, indicated as black and green circles. (b) In another pathway, DDR1b assembles into clusters within minutes of collagen I stimulation (likely binding to non-fibrillar collagen present at early stages of fibrillogenesis) and gets endocytosed. However, phosphorylation at Y513 and Y792 is not detected in these clusters at the early (~30 min) time point. At later times (~4 h) post- ligand administration, phosphorylation of DDR1b in its IJXM (Y513) (green circles) but not in its KD (Y792) is detected in the DDR1b clusters. At present, it is not clear if Y513-positive clusters are present on the cell surface or constitute pools of endocytosed DDR1b receptors localizing within endosomes. Owing to the fact that DDRs contain numerous Tyr residues within the IJXM region and their KDs, phosphorylation at other Tyr residues (indicated by the red dashed circles) may also be ensuing in the structures described in panels a and b but are undetectable by the tools available at this time. It is likely that formation of higher-order assemblies of DDRs may recruit additional cytosolic proteins, which mediate specific cellular processes.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513, catalog no. 14531), monoclonal antibody recognizing total DDR1 (D1G6, catalog no. 5583), polyclonal antibody recognizing phosphoDDR1 (DDR1-Y792, catalog no.11994), polyclonal antibody recognizing total DDR2 (catalog no.12133) andDyLightTM 554 Phalloidin (catalog no. 13054) were all purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Phospho-proteomics, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Two-step release of kinase autoinhibition in discoidin domain receptor 1

doi: 10.1073/pnas.2007271117

Figure Lengend Snippet: The DDR1 kinase is autoinhibited by the juxtamembrane region, JM4. (A) Domain organization of DDR1. DS, discoidin domain; DS-like, discoidin-like domain; TM, transmembrane region; JM4, juxtamembrane region 4. The plasma membrane is depicted as a horizontal bar in light gray. Selected tyrosine residues are indicated by open circles and labeled. The A-loop of the kinase domain (green) contains a total of three tyrosines (Tyr792, Tyr796, Tyr797). (B) Sequence alignment of the JM4 regions of human DDR1 and DDR2. The two JM4 tyrosines, Tyr569 and Tyr586, are highlighted. (C) Full-length DDR1b, wild type, or indicated mutants, were transiently expressed in HEK293 cells. Cells were stimulated with collagen I (+) or left unstimulated (−) for 90 min at 37 °C. Aliquots of cell lysates were analyzed by SDS/PAGE and Western blotting with the A-loop-specific anti-phosphotyrosine antibody, anti-pY796. Total DDR1 was then detected using anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left. (D) Crystal structure of the DDR1 JM4-kinase Y569F/Y586F mutant. The crystallographically resolved part of the JM4 region (residues 574 to 601) is in orange and the A-loop is in green. The side chain of Phe586 is shown in atomic detail. Disordered loop regions are shown with dotted lines, and the putative position of Phe569 (see text) is indicated. (E) Close-up view of the JM4 region inserted into the active site of the DDR1 kinase. Selected side chains are shown in atomic detail and labeled. The color scheme is the same as in D.

Article Snippet: The following DDR1-specific anti-phosphotyrosine antibodies were used to determine DDR1 phosphorylation: rabbit anti-phospho-DDR1-pY796 (R&D Systems; MAB25382, lot CJCT0217081), rabbit anti-phospho-DDR1-pY569 (Biomatik, custom-made), and rabbit anti-phospho-DDR1-pY586 (Biomatik, custom-made).

Techniques: Labeling, Sequencing, SDS Page, Western Blot, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Two-step release of kinase autoinhibition in discoidin domain receptor 1

doi: 10.1073/pnas.2007271117

Figure Lengend Snippet: The DDR1 JM4 region, but not the A-loop, of soluble kinase constructs is autophosphorylated at low protein concentration. The soluble DDR1 kinase constructs, JM4K-WT, JM4K-Y569F/Y586F, and kinase alone, were incubated at 1-µM concentration with 1 mM ATP for the indicated times (0 to 120 min) at 20 °C. Protein samples were then boiled in sample buffer and analyzed by SDS/PAGE and Western blotting with a pan-anti-phosphotyrosine antibody (anti-pY), JM4-specific (anti-pY569, anti-pY586), or A-loop-specific (anti-pY796), anti-phosphotyrosine antibodies. Total DDR1 was then detected with anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left.

Article Snippet: The following DDR1-specific anti-phosphotyrosine antibodies were used to determine DDR1 phosphorylation: rabbit anti-phospho-DDR1-pY796 (R&D Systems; MAB25382, lot CJCT0217081), rabbit anti-phospho-DDR1-pY569 (Biomatik, custom-made), and rabbit anti-phospho-DDR1-pY586 (Biomatik, custom-made).

Techniques: Construct, Protein Concentration, Incubation, Concentration Assay, SDS Page, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Two-step release of kinase autoinhibition in discoidin domain receptor 1

doi: 10.1073/pnas.2007271117

Figure Lengend Snippet: Autophosphorylation of soluble DDR1 JM4-kinase occurs in two distinct steps. The soluble DDR1 kinase constructs, JM4K-WT, JM4K-Y569F/Y586F, and kinase alone, were incubated at 100-µM concentration in the presence of 20 mM ATP for the indicated times (0 to 180 min) at 20 °C. (A) Protein samples were then separated by native PAGE and the gel was stained with InstantBlue Coomassie. (B) Aliquots of the same samples were boiled in sample buffer and analyzed by SDS/PAGE and Western blotting with JM4-specific (anti-pY569, anti-pY586) or A-loop-specific (anti-pY796) anti-phosphotyrosine antibodies. Total DDR1 was then detected with anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left.

Article Snippet: The following DDR1-specific anti-phosphotyrosine antibodies were used to determine DDR1 phosphorylation: rabbit anti-phospho-DDR1-pY796 (R&D Systems; MAB25382, lot CJCT0217081), rabbit anti-phospho-DDR1-pY569 (Biomatik, custom-made), and rabbit anti-phospho-DDR1-pY586 (Biomatik, custom-made).

Techniques: Construct, Incubation, Concentration Assay, Clear Native PAGE, Staining, SDS Page, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Two-step release of kinase autoinhibition in discoidin domain receptor 1

doi: 10.1073/pnas.2007271117

Figure Lengend Snippet: JM4 and A-loop phosphorylation sequentially increase the enzymatic activity of DDR1 kinase. (A) JM4-kinase was incubated at 100-µM concentration in the presence of 20 mM ATP for 90 min at 20 °C. The product was then dialyzed into 80 mM NaCl, 50 mM Tris pH 8.5 and separated by ion exchange chromatography (IEX). Protein was collected in the flow-through (FT) and in two peaks eluted with a NaCl gradient (E1 and E2). (B) The unphosphorylated protein (JM4K-0P), IEX input, and IEX fractions from A were separated by native PAGE and stained with InstantBlue Coomassie. 0P, position of unphosphorylated JM4K; IP, position of JM4K in an intermediate phosphorylation state; FP, position of fully phosphorylated JM4K. (C) The different phosphoforms of JM4-kinase obtained by IEX in A, as well as unphosphorylated (kinase-0P) and phosphorylated kinase alone (kinase-P) were boiled in sample buffer and separated by SDS/PAGE. Samples were then analyzed by Western blotting with a pan-anti-phosphotyrosine antibody (anti-pY), as well as with JM4-specific (anti-pY569, anti-pY586) or A-loop-specific (anti-pY796) anti-phosphotyrosine antibodies, before total DDR1 detection using anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left. (D) In vitro kinase activity of the different phosphoforms of JM4-kinase and kinase alone using Axltide peptide as a substrate. Initial velocities were determined over a range of ATP concentrations in the presence of saturating peptide (515 µM; Upper graph) or over a range of peptide concentrations in the presence of saturating ATP (800 µM; Lower graph). Data were fitted with the Michaelis–Menten equation (n = 3; mean and SEM shown). SA, specific activity; JM4K-0P, unphosphorylated JM4K; JM4K-IP, JM4K in the intermediate phosphorylation state; JM4K-FP, fully phosphorylated JM4K; K-0P, unphosphorylated kinase; and K-P, fully phosphorylated kinase.

Article Snippet: The following DDR1-specific anti-phosphotyrosine antibodies were used to determine DDR1 phosphorylation: rabbit anti-phospho-DDR1-pY796 (R&D Systems; MAB25382, lot CJCT0217081), rabbit anti-phospho-DDR1-pY569 (Biomatik, custom-made), and rabbit anti-phospho-DDR1-pY586 (Biomatik, custom-made).

Techniques: Activity Assay, Incubation, Concentration Assay, Ion Exchange Chromatography, Clear Native PAGE, Staining, SDS Page, Western Blot, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Two-step release of kinase autoinhibition in discoidin domain receptor 1

doi: 10.1073/pnas.2007271117

Figure Lengend Snippet: Phosphomimetic mutation relieves JM4 autoinhibition. (A) In vitro kinase activity, using Axltide peptide as a substrate, of unphosphorylated JM4-kinase (JM4K-WT-0P), JM4-phosphorylated JM4-kinase (JM4K-WT-IP), unphosphorylated JM4-kinase Y569E/Y586E mutant (JM4K-YE-0P), and unphosphorylated kinase (K-0P). Initial velocities were determined over a range of ATP concentrations in the presence of saturating peptide (515 µM; Upper graph) or over a range of peptide concentrations in the presence of saturating ATP (800 µM; Lower graph). Data were fitted with the Michaelis–Menten equation (n = 3; mean and SEM shown). SA, specific activity. (B) Autophosphorylation kinetics of soluble DDR1 kinase constructs. The indicated soluble kinase constructs were incubated at 100-µM concentration in the presence of 20 mM ATP for the indicated time (0 to 180 min) at 20 °C. Protein samples were then separated by native PAGE and the gel was stained with InstantBlue Coomassie. (C) The same samples shown in A were boiled in sample buffer and separated by SDS/PAGE. Samples were then analyzed by Western blotting with the A-loop-specific anti-phosphotyrosine antibody, anti-pY796. Total DDR1 was then detected with anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left.

Article Snippet: The following DDR1-specific anti-phosphotyrosine antibodies were used to determine DDR1 phosphorylation: rabbit anti-phospho-DDR1-pY796 (R&D Systems; MAB25382, lot CJCT0217081), rabbit anti-phospho-DDR1-pY569 (Biomatik, custom-made), and rabbit anti-phospho-DDR1-pY586 (Biomatik, custom-made).

Techniques: Mutagenesis, In Vitro, Activity Assay, Construct, Incubation, Concentration Assay, Clear Native PAGE, Staining, SDS Page, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Two-step release of kinase autoinhibition in discoidin domain receptor 1

doi: 10.1073/pnas.2007271117

Figure Lengend Snippet: Autophosphorylation in vitro of full-length DDR1. (A) Full-length DDR1b wild type or the indicated DDR1 mutants were transiently expressed in HEK293 cells (ΔJM4, deletion of JM4 region). DDR1 was immunoprecipitated from cell lysates, and immunoprecipitates were incubated in the presence (+) or absence (−) of 1 mM ATP for 30 min at 30 °C. Samples were then boiled in sample buffer and analyzed by SDS/PAGE and Western blotting with the A-loop-specific anti-phosphotyrosine antibody, anti-pY796. Total DDR1 was then detected with anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left (Upper). The Lower shows the quantification of the anti-pY796 signals, normalized to respective anti-DDR1 signals. The anti-pY796 signal is expressed as a percentage of the sum of all bands on a blot, with the mean and SEM shown (n = 4). Statistically significant differences (P < 0.0001) between the +ATP signals for DDR1 wild type and the Y569F/Y586F mutant are marked by four asterisks (****). The +ATP signals for the Y569E/Y586E and ΔJM4 mutants were not significantly different from that of wild-type DDR1 (P = 0.7113, DDR1-Y569E/Y586E vs. DDR1-WT; P > 0.999, DDR1-ΔJM4 vs. DDR1-WT; two-way ANOVA with Tukey’s post hoc test). (B) Full-length wild-type DDR1b was transiently expressed in HEK293 cells before being immunoprecipitated, as in A and stimulated with 1 mM ATP for the indicated time course (0 to 60 min). Samples were then boiled in sample buffer and analyzed by SDS/PAGE and Western blotting with JM4-specific (anti-pY569, anti-pY586) or A-loop-specific (anti-pY796) anti-phosphotyrosine antibodies. Total DDR1 was then detected with anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left. The Bottom shows the quantification of anti-phospho-DDR1 signals relative to respective DDR1 signals, expressed as in A (n = 3). Statistically significant differences (P < 0.01) between the anti-pY569 and anti-pY796 signals are indicated by double asterisks (**P = 0.0072, t = 10 min; P = 0.0032, t = 20 min; P = 0.0034, t = 60 min; two-way ANOVA with Tukey’s post hoc test). (C) The full-length DDR1 constructs used in A were transiently expressed in HEK293 cells. Cells were stimulated with collagen I (+) or left unstimulated (−) for 90 min at 37 °C. Aliquots of cell lysates were analyzed by SDS/PAGE and Western blotting with the A-loop-specific anti-phosphotyrosine antibody, anti-pY796. Total DDR1 was then detected with anti-DDR1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the Left.

Article Snippet: The following DDR1-specific anti-phosphotyrosine antibodies were used to determine DDR1 phosphorylation: rabbit anti-phospho-DDR1-pY796 (R&D Systems; MAB25382, lot CJCT0217081), rabbit anti-phospho-DDR1-pY569 (Biomatik, custom-made), and rabbit anti-phospho-DDR1-pY586 (Biomatik, custom-made).

Techniques: In Vitro, Immunoprecipitation, Incubation, SDS Page, Western Blot, Mutagenesis, Construct

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