Journal: Oncology reports

Article Title: DDR1 promotes breast tumor growth by suppressing antitumor immunity.

doi: 10.3892/or.2019.7338

Figure Lengend Snippet: Figure 1. Upregulation of DDR1 in breast cancer. (A) Reverse transcription‑quantitative PCR was used to examine the mRNA levels of DDR1 in breast cancer tissues compared with adjacent non‑tumor tissues from 20 patients enrolled in the present study. (B and C) Mean expression levels of DDR1 in TCGA breast cancer datasets. (D) The protein expression levels of DDR1 were detected by western blot analysis in 10 pairs of breast cancer tissues and adjacent non‑tumor tissues. Values are presented as the mean ± standard error of the mean. DDR1, discoidin domain receptor tyrosine kinase 1; TCGA, The Cancer Genome Atlas; HER2, epidermal growth factor receptor 2; N, normal; T, tumor.

Article Snippet: Membranes were incubated with the following primary antibodies: DDR1 (1:1,000; cat. no. sc-532; Santa Cruz Biotechnology, Inc.), DDR1 (1:1,000; cat. no. AF2396; R&D Systems, Inc.), and β‐actin (1:10,000; cat. no. sc‐47778; Santa Cruz Biotechnology, Inc.), followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. nos.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: DDR1 promotes breast tumor growth by suppressing antitumor immunity.

doi: 10.3892/or.2019.7338

Figure Lengend Snippet: Figure 2. Ectopic expression of DDR1 promotes tumor growth in vivo. (A) Western blot analysis of DDR1 protein expression levels following stable overexpres- sion of DDR1 in 4T1 cells compared with vector control. (B) In vitro cell proliferation assay was performed with 4T1 vector control and DDR1‑overexpressing cells. (C) In vivo tumorigenesis assay was performed with the 4T1 vector control and DDR1‑overexpressing 4T1 cells. Tumor volumes and (D) photographs are shown (n=5 per group). (E) Flow cytometry analysis of dissociated tumors (n=5 per group) for CD4+ and (F) CD8+ infiltrating T cells. DDR1, discoidin domain receptor tyrosine kinase 1; OD, optical density.

Article Snippet: Membranes were incubated with the following primary antibodies: DDR1 (1:1,000; cat. no. sc-532; Santa Cruz Biotechnology, Inc.), DDR1 (1:1,000; cat. no. AF2396; R&D Systems, Inc.), and β‐actin (1:10,000; cat. no. sc‐47778; Santa Cruz Biotechnology, Inc.), followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. nos.

Techniques: Expressing, In Vivo, Western Blot, Plasmid Preparation, Control, In Vitro, Proliferation Assay, Flow Cytometry

Journal: Oncology reports

Article Title: DDR1 promotes breast tumor growth by suppressing antitumor immunity.

doi: 10.3892/or.2019.7338

Figure Lengend Snippet: Figure 3. Deletion of DDR1 inhibits tumor growth in vivo. (A) Establishment of DDR1 KO EMT6 cell lines using the CRISPR/Cas9 method. Western blotting revealed that DDR1 expression was obviously depleted in DDR1 KO1 and KO2 clones. (B) DNA and protein sequences of control and DDR1 KO EMT6 cell clones. (C) There was no significant difference observed by cell proliferation assay in vitro between the DDR1 KO EMT6 cells and the control cells. (D and E) DDR1 KO significantly decreased tumor growth compared with the control group in vivo (n=4 per group). DDR1, discoidin domain receptor tyrosine kinase 1; KO, knockout; OD, optical density.

Article Snippet: Membranes were incubated with the following primary antibodies: DDR1 (1:1,000; cat. no. sc-532; Santa Cruz Biotechnology, Inc.), DDR1 (1:1,000; cat. no. AF2396; R&D Systems, Inc.), and β‐actin (1:10,000; cat. no. sc‐47778; Santa Cruz Biotechnology, Inc.), followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. nos.

Techniques: In Vivo, CRISPR, Western Blot, Expressing, Clone Assay, Control, Proliferation Assay, In Vitro, Knock-Out

Journal: Oncology reports

Article Title: DDR1 promotes breast tumor growth by suppressing antitumor immunity.

doi: 10.3892/or.2019.7338

Figure Lengend Snippet: Figure 4. Deletion of DDR1 elicits a distinct immunophenotypic signature in EMT6 tumor‑bearing mice. Flow cytometry analysis of dissociated tumors (n=4 per group) for the percentage of (A) CD4+ T cells, (B) CD8+ T cells, (C) early activated CD8+ T cells, (D) proliferative CD8+ T cells, and (E) T‑bet+ CD8+ infiltrating T cells. DDR1, discoidin domain receptor tyrosine kinase 1; T‑bet, T‑box transcription factor 21; KO, knockout.

Article Snippet: Membranes were incubated with the following primary antibodies: DDR1 (1:1,000; cat. no. sc-532; Santa Cruz Biotechnology, Inc.), DDR1 (1:1,000; cat. no. AF2396; R&D Systems, Inc.), and β‐actin (1:10,000; cat. no. sc‐47778; Santa Cruz Biotechnology, Inc.), followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. nos.

Techniques: Flow Cytometry, Knock-Out

Journal: Oncology reports

Article Title: DDR1 promotes breast tumor growth by suppressing antitumor immunity.

doi: 10.3892/or.2019.7338

Figure Lengend Snippet: Figure 6. Correlation between DDR1 expression and immunity in TCGA breast cancer cohort. (A) Correlation between DDR1 mRNA expression levels and tumor purity (cor=0.184, P=5.16x10‑9), CD4+ T cell count (cor=‑0.221, P=2.70x10‑12) and CD8+ T cell count (cor=‑0.107, P=8.85x10‑4). (B) Correlation between DDR1 mRNA expression levels and tumor‑infiltrating lymphocyte signature genes CD4 (cor=‑0.265, P=5.99x10‑19) and CD8A (cor=‑0.201, P=1.97x10‑11), as well as the cytotoxic T cell marker GZMB (cor=‑0.208, P=3.22x10‑12). DDR1, discoidin domain receptor tyrosine kinase 1; TCGA, The Cancer Genome Atlas; GZMB, granzyme B; BRCA, breast cancer.

Article Snippet: Membranes were incubated with the following primary antibodies: DDR1 (1:1,000; cat. no. sc-532; Santa Cruz Biotechnology, Inc.), DDR1 (1:1,000; cat. no. AF2396; R&D Systems, Inc.), and β‐actin (1:10,000; cat. no. sc‐47778; Santa Cruz Biotechnology, Inc.), followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. nos.

Techniques: Expressing, Cell Counting, Marker

Journal: Oncology reports

Article Title: DDR1 promotes breast tumor growth by suppressing antitumor immunity.

doi: 10.3892/or.2019.7338

Figure Lengend Snippet: Figure 5. Effects of ECD neutralizing antibody on breast tumor growth in vivo. (A) Secreted DDR1‑ECD protein was detected in the conditioned media of EMT6 cells by western blot analysis. (B and C) Tumor volumes and photographs of EMT6 cells in BALB/c nude mice treated with DR1‑ECD neutralizing antibody or control IgG (n=5 per group). (D) Flow cytometry analysis of dissociated tumors (n=5 per group) for the percentage of CD4+ and (E) CD8+ T cells. ECD, extracellular domain; DDR1, discoidin domain receptor tyrosine kinase 1; IgG, immunoglobulin G; KO, knockout.

Article Snippet: Membranes were incubated with the following primary antibodies: DDR1 (1:1,000; cat. no. sc-532; Santa Cruz Biotechnology, Inc.), DDR1 (1:1,000; cat. no. AF2396; R&D Systems, Inc.), and β‐actin (1:10,000; cat. no. sc‐47778; Santa Cruz Biotechnology, Inc.), followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. nos.

Techniques: In Vivo, Western Blot, Control, Flow Cytometry, Knock-Out

Journal: Journal of Drug Targeting

Article Title: Antibody-drug conjugates targeting DDR1 as a novel strategy for treatment of breast cancer

doi: 10.1080/1061186x.2024.2386621

Figure Lengend Snippet: Figure 1. DDR1 Expression in breast cancer. a representative IHC images of DDR1 expression in 140 breast cancer tissues. a. Samples were stained for the DDR1 antigen, visualised as brown staining in the membrane or cytoplasm, and counterstained with haematoxylin, resulting in blue staining in the nucleus. Representative images indicate staining intensity levels: 0 for no detectable staining, 1+ for weak staining, 2+ for moderate staining, and 3+ for strong staining. The magnifications are 1x, 4x and 8x. b. Stacked bar plot showing the distribution of low and high DDR1 expression in 140 breast cancer tissues classified by clinical stage. c. Box plot comparing the RNA expression level of DDR1 in normal and breast cancer samples. RNA expression levels are increased in breast cancer samples in the cancer genome atlas (TCGA) database. The red column indicates tumour tissues, and the black column indicates normal tissues. ns: no significance.

Article Snippet: Immunohistochemistry (IHC) staining of DDR1 with tissue microarray (TMA) to evaluate DDR1 expression, tMa composed of small circular samples of paraffin-embedded cancerous tissue from 140 patients with different stages of Bc was used (shanghai Outdo Biotech, china). ihc staining was performed to detect DDR1 expression referred to our previously published methods [9]. a 1:100 dilution of primary antibody against DDR1 from Novus Biologicals (colorado, Usa) was used. the percentage of positive cells and the strength of staining codetermined the staining intensity. intensity scores were defined as follows: negative, no detectable staining signal in > 50% of tumour cells; 1 + intensity, weak staining signal detected in > 50% of tumour cells; 2+ intensity, moderate staining signal in > 50% of tumour cells; and 3+ intensity, strong staining signal in > 50% of tumour cells. the staining results were further dichotomised into low DDR1 expression (negative/1+ intensity scores) and high DDR1 expression (2+/3+ intensity scores). two pathologists who were unaware of the clinicopathological variables reviewed and graded the ihc staining intensity of the tMas.

Techniques: Expressing, Staining, Membrane, RNA Expression

Journal: Journal of Drug Targeting

Article Title: Antibody-drug conjugates targeting DDR1 as a novel strategy for treatment of breast cancer

doi: 10.1080/1061186x.2024.2386621

Figure Lengend Snippet: Figure 2. FCM histograms of DDR1 cell surface expression in breast cancer cell lines. Cells were detected with an isotype mAb (blue) or anti-DDR1 mAb (red), respectively. Numbers represent the percentages of DDR1-positive cells [13]. Samples were analysed using NovoExpress software.

Article Snippet: Immunohistochemistry (IHC) staining of DDR1 with tissue microarray (TMA) to evaluate DDR1 expression, tMa composed of small circular samples of paraffin-embedded cancerous tissue from 140 patients with different stages of Bc was used (shanghai Outdo Biotech, china). ihc staining was performed to detect DDR1 expression referred to our previously published methods [9]. a 1:100 dilution of primary antibody against DDR1 from Novus Biologicals (colorado, Usa) was used. the percentage of positive cells and the strength of staining codetermined the staining intensity. intensity scores were defined as follows: negative, no detectable staining signal in > 50% of tumour cells; 1 + intensity, weak staining signal detected in > 50% of tumour cells; 2+ intensity, moderate staining signal in > 50% of tumour cells; and 3+ intensity, strong staining signal in > 50% of tumour cells. the staining results were further dichotomised into low DDR1 expression (negative/1+ intensity scores) and high DDR1 expression (2+/3+ intensity scores). two pathologists who were unaware of the clinicopathological variables reviewed and graded the ihc staining intensity of the tMas.

Techniques: Expressing, Software

Journal: Journal of Drug Targeting

Article Title: Antibody-drug conjugates targeting DDR1 as a novel strategy for treatment of breast cancer

doi: 10.1080/1061186x.2024.2386621

Figure Lengend Snippet: Figure 3. Structure and characterisation of T4H11-DM4. a. Chemical structure and mechanism diagram of T4H11-DM4. T4H11-DM4 contains three key components, an anti-DDR1 antibody named T4H11, a cleavable linker SPDB, and a microtubule inhibitor payload DM4. SPDB-DM4 is attached to the amino group of the antibody lysine. The major mechanism of action of T4H11-DM4 involves the following steps: ① binding of T4H11-DM4 to membrane-bound DDR1; ② internalisation of T4H11-DM4 by DDR1-mediated endocytosis; fusion of endosomes with lysosomes; ③ degradation of T4H11-DM4 and release of the cytotoxic payload DM4; ④ inhibition of cell mitosis; and ⑤ induction of tumour cell apoptosis. b. FCM analysis was performed to assess the binding and internalisation ability of T4H11 and T4H11-DM4. To assess binding ability, ZR-75-1 cells were incubated with an isotype mAb (blue), T4H11 (red), or T4H11-DM4 (green) at 4 °C. For internalisation ability, yellow peaks represent T4H11 or T4H11-DM4 shifted to 37 °C for 3 h. The internalisation rates of T4H11 and T4H11-DM4 in ZR-75-1 cells were 64% and 67%, respectively. c. In vivo distribution of T4H11 in ZR-75-1 tumour-bearing mice at 2 and 4 hours after a single intravenous administration of the isotype mAb-Cy5.5 (left) and T4H11-Cy5.5 (right), respectively. T4H11-Cy5.5 could specifically target tumour tissue at 2 h post injection and the fluorescence intensity in tumour was increased at 6 h post injection. Isotype mAb-Cy5.5 distributed sporadically and not concentrated in tumour tissues. The tumour tissue areas were circled and pointed with arrows, specifically mAb-Cy5.5 (pink circle and arrow) and T4H11-Cy5.5 (purple circle and arrow).

Article Snippet: Immunohistochemistry (IHC) staining of DDR1 with tissue microarray (TMA) to evaluate DDR1 expression, tMa composed of small circular samples of paraffin-embedded cancerous tissue from 140 patients with different stages of Bc was used (shanghai Outdo Biotech, china). ihc staining was performed to detect DDR1 expression referred to our previously published methods [9]. a 1:100 dilution of primary antibody against DDR1 from Novus Biologicals (colorado, Usa) was used. the percentage of positive cells and the strength of staining codetermined the staining intensity. intensity scores were defined as follows: negative, no detectable staining signal in > 50% of tumour cells; 1 + intensity, weak staining signal detected in > 50% of tumour cells; 2+ intensity, moderate staining signal in > 50% of tumour cells; and 3+ intensity, strong staining signal in > 50% of tumour cells. the staining results were further dichotomised into low DDR1 expression (negative/1+ intensity scores) and high DDR1 expression (2+/3+ intensity scores). two pathologists who were unaware of the clinicopathological variables reviewed and graded the ihc staining intensity of the tMas.

Techniques: Binding Assay, Membrane, Inhibition, Incubation, In Vivo, Injection, Fluorescence

Journal: Journal of Drug Targeting

Article Title: Antibody-drug conjugates targeting DDR1 as a novel strategy for treatment of breast cancer

doi: 10.1080/1061186x.2024.2386621

Figure Lengend Snippet: Figure 4. In vitro cytotoxicity mediated by the anti-DDR1 ADC in breast cancer cell lines. Cell viability was assessed after 72 h of exposure to T4H11 (solid square; black) or T4H11-DM4 (solid circle; red) at various concentrations using CCK-8 assay.

Article Snippet: Immunohistochemistry (IHC) staining of DDR1 with tissue microarray (TMA) to evaluate DDR1 expression, tMa composed of small circular samples of paraffin-embedded cancerous tissue from 140 patients with different stages of Bc was used (shanghai Outdo Biotech, china). ihc staining was performed to detect DDR1 expression referred to our previously published methods [9]. a 1:100 dilution of primary antibody against DDR1 from Novus Biologicals (colorado, Usa) was used. the percentage of positive cells and the strength of staining codetermined the staining intensity. intensity scores were defined as follows: negative, no detectable staining signal in > 50% of tumour cells; 1 + intensity, weak staining signal detected in > 50% of tumour cells; 2+ intensity, moderate staining signal in > 50% of tumour cells; and 3+ intensity, strong staining signal in > 50% of tumour cells. the staining results were further dichotomised into low DDR1 expression (negative/1+ intensity scores) and high DDR1 expression (2+/3+ intensity scores). two pathologists who were unaware of the clinicopathological variables reviewed and graded the ihc staining intensity of the tMas.

Techniques: In Vitro, CCK-8 Assay

Journal: Journal of Drug Targeting

Article Title: Antibody-drug conjugates targeting DDR1 as a novel strategy for treatment of breast cancer

doi: 10.1080/1061186x.2024.2386621

Figure Lengend Snippet: Figure 5. In vivo antitumor activity of DDR1-DM4 in breast cancer. a. Tumour growth curves for the ZR-75-1 and T47D xenograft models treated with 2.5, 5, or 10 mg/kg T4H11-DM4 for three injections. Xenograft BALB/c nude mice (ZR-75-1) or NOD-SCID mice (T47D) were intravenously treated with 2.5, 5, or 10 mg/kg T4H11-DM4, 10 mg/kg T4H11 or vehicle (PBS) via a q3d × 3 regimen. b. Tumour growth curve for ZR-75-1 xenograft mice treated with 2.5, 5, or 10 mg/kg T4H11-DM4 for one injection. Tumour growth studies were performed as indicated (arrow). The error bars represent SD of the mean. The individual tumour volumes and statistical analysis on the last day of observation period in every model were shown in column graphs. ***p < 0.001, **p < 0.01, *p < 0.05, ns: no significance.

Article Snippet: Immunohistochemistry (IHC) staining of DDR1 with tissue microarray (TMA) to evaluate DDR1 expression, tMa composed of small circular samples of paraffin-embedded cancerous tissue from 140 patients with different stages of Bc was used (shanghai Outdo Biotech, china). ihc staining was performed to detect DDR1 expression referred to our previously published methods [9]. a 1:100 dilution of primary antibody against DDR1 from Novus Biologicals (colorado, Usa) was used. the percentage of positive cells and the strength of staining codetermined the staining intensity. intensity scores were defined as follows: negative, no detectable staining signal in > 50% of tumour cells; 1 + intensity, weak staining signal detected in > 50% of tumour cells; 2+ intensity, moderate staining signal in > 50% of tumour cells; and 3+ intensity, strong staining signal in > 50% of tumour cells. the staining results were further dichotomised into low DDR1 expression (negative/1+ intensity scores) and high DDR1 expression (2+/3+ intensity scores). two pathologists who were unaware of the clinicopathological variables reviewed and graded the ihc staining intensity of the tMas.

Techniques: In Vivo, Activity Assay, Injection

Journal: Iranian Biomedical Journal

Article Title: Evaluation of t-DARPP Expression Alteration in Association with DDR1 Expression in Non-Small Cell Lung Cancer

doi: 10.61186/ibj.3878

Figure Lengend Snippet: Uniqueness and authenticity of the RT-PCR products. (A) The size and uniqueness of DDR1, t-DARPP, and GAPDH PCR products following agarose gel electrophoresis. (B) The authenticity of the PCR amplified products shown by single, sharp melting curves

Article Snippet: Next, the membranes were incubated overnight with primary goat anti-DDR1 antibodies (1:1,000 dilution; AF2396, R&D Systems, Minneapolis, USA) or mouse anti-β-actin antibodies (1:5,000 dilution; MABT825, MilliporeSigma, Burlington, USA) in 2.5% BSA in TBS containing 0.1% Tween-20 at 4 °C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification

Journal: Iranian Biomedical Journal

Article Title: Evaluation of t-DARPP Expression Alteration in Association with DDR1 Expression in Non-Small Cell Lung Cancer

doi: 10.61186/ibj.3878

Figure Lengend Snippet: Expression of DDR1 after collagen type I simulation and siRNA transfection in A549 and Calu-3 cells. (A) Expression analysis of DDR1 mRNA in the cells incubated with collagen type I at various times by RT-PCR. DDR1 mRNA expressions increased significantly after 16 hours. The mean fold change of DDR1 expression was calculated relative to unstimulated controls. (B) Western blot analysis of DDR1 protein expression in A549 and Calu-3 cells incubated with collagen type I and fibronectin as control after 16 h; DDR1 expression enhanced under the collagen type I stimulation. (C) Analysis of DDR1 expression after siRNA transfection in cells using RT-PCR. DDR1 mRNA expressions decreased significantly after si-DDR1 transfection compared to non-targeting control siRNA. All experiments were conducted in three biological and two technical replicates ( * p < 0.05)

Article Snippet: Next, the membranes were incubated overnight with primary goat anti-DDR1 antibodies (1:1,000 dilution; AF2396, R&D Systems, Minneapolis, USA) or mouse anti-β-actin antibodies (1:5,000 dilution; MABT825, MilliporeSigma, Burlington, USA) in 2.5% BSA in TBS containing 0.1% Tween-20 at 4 °C.

Techniques: Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Iranian Biomedical Journal

Article Title: Evaluation of t-DARPP Expression Alteration in Association with DDR1 Expression in Non-Small Cell Lung Cancer

doi: 10.61186/ibj.3878

Figure Lengend Snippet: Analysis of t-DARPP expression in A549 and Calu-3 cells after induction by collagen type I and knockdown DDR1. RT-PCR was conducted to evaluate the t-DARPP expression in NSCLC cells stimulated by collagen type I in a time-dependent manner after DDR1 siRNA transfection. (A) Relative t-DARPP mRNA expressions significantly increased in the cells after 16 hours. The mean fold change of t-DARPP expression was calculated relative to unstimulated controls. (B) Relative t-DARPP mRNA expressions significantly decreased after si-DDR1 transfection versus non-targeting control siRNA in cells. All experiments were conducted in three biological and two technical replicates ( * p < 0.05)

Article Snippet: Next, the membranes were incubated overnight with primary goat anti-DDR1 antibodies (1:1,000 dilution; AF2396, R&D Systems, Minneapolis, USA) or mouse anti-β-actin antibodies (1:5,000 dilution; MABT825, MilliporeSigma, Burlington, USA) in 2.5% BSA in TBS containing 0.1% Tween-20 at 4 °C.

Techniques: Expressing, Knockdown, Reverse Transcription Polymerase Chain Reaction, Transfection, Control

Journal: Science (New York, N.Y.)

Article Title: The target landscape of clinical kinase drugs

doi: 10.1126/science.aan4368

Figure Lengend Snippet: Characterization of drugs targeting MELK. (A) Kinobeads profiling of 15 non-small cell lung cancer (NSCLC) patient tumours and adjacent healthy tissue identified over-expression of MELK, EGFR and DDR1 in the tumours. Expression was confirmed by immunohistochemistry in a cohort of 375 NSCLC patients. (B) MELK over-expression correlated (log rank test) with poor overall survival in squamous cell carcinoma (SCC) but not adenocarcinoma (ADC). Therefore, MELK may have potential as a predictive survival marker in SCC. (C) Radar plot depicting targets and binding affinities of the designated phase I MELK inhibitor OTS-167 (each spoke is a direct binder and the length of the spoke is indicative of binding affinity) showing that the drug is a very unselective compound and suggesting that its biological activities may not be due to MELK inhibition alone. MELK is marked by a red dot. (D) Kinase activity assays confirmed that MELK binders identified in this study (e.g., Nintedanib) are also potent MELK inhibitors. (E) Co-crystal structures obtained for five MELK inhibitors revealed that e.g., Nintedanib forms strong interactions with residues E15 and E57 in the ATP pocket. There are additional residues (notably C70 and C89) that may be exploited to develop selective and potent irreversible MELK inhibitors. Further details are provided in Figure S9 and the Supplementary Materials.

Article Snippet: Immunohistochemical staining was performed on a Discovery XT automated stainer (Ventana) using the following primary antibodies: MELK (1:50, Sigma-Aldrich), DDR1 (1:50, Biozol), and EGFR (pharmDx™-kit, DAKO).

Techniques: Over Expression, Expressing, Immunohistochemistry, Marker, Binding Assay, Inhibition, Activity Assay

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: A Schematic diagram of parallel-plate flow chamber. B Left: Immunofluorescence of F-actin and DDR1 in HUVECs that were transfected with DDR1-siRNA or scrambled control siRNA (si-Scr) and subjected to PS or OS for 24 h. HUVECs were all grown on gelatin-coated glass slide. Right: Quantification of alignment by measuring the orientation angle. n = 9 images from 3 biological replicates. Scale bar: 50 μm. C Left: Immunofluorescence of F-actin and DDR1 in NIH3T3 cells that were infected with DDR1-EGFP adenovirus and subjected to PS or OS for 24 h. Right: Quantification of alignment by measuring the orientation angle. n = 9 images from 3 biological replicates. Scale bar: 50 μm. D Quantitative RT-PCR (qRT-PCR) to detect anti-inflammatory transcription factors (KLF2 and KLF4) and proinflammatory genes (MCP1 and E-selectin) in HUVECs treated as in ( B ). Data were analyzed by two-way ANOVA followed by Tukey’s multiply test. E Schematic diagram of experimental design. F Representative gross images of carotid arteries from the indicated mice at 4 weeks after partial ligation. Scale bar: 2 mm. 3 different positions of carotid artery are showed, and the definitions of upper, middle, and lower are consistent with anatomical position. G Representative H&E and Oil red O staining of neointima in the left common carotid arteries from the Ddr1 WT and Ddr1 iECKO mice at 4wk after ligation. Scale bar: 200 μm. H Quantification of the atherosclerotic lesion area. n = 7 mice. The most severe atherosclerotic lesion of the left common carotid artery was counted. Data were analyzed by two-way ANOVA followed by Tukey’s multiply test. I Representative Immunofluorescence staining of E-selectin, ICAM1, and VCAM1 in cross-sections of the left common carotid arteries from the Ddr1 WT and Ddr1 iECKO mice at 1 week post-operation. Scale bar: 50 μm. Data were expressed as the means ± SEM.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Immunofluorescence, Transfection, Control, Infection, Quantitative RT-PCR, Ligation, Staining

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: A Representative immunofluorescence of DDR1 in HUVECs subjected to PS or OS for six time periods (15 min, 30 min, 1 h, 3 h, 6 h and 24 h). HUVECs were grown on gelatin-coated glass slide. B Quantification of DDR1 condensates in ( A ), n = 60 images from 6 biological replicates. Data were expressed as the means ± SD. C Non-reducing SDS-PAGE to detect DDR1 oligomerization in HUVECs subjected to PS or OS for indicated time. D Quantification of the fraction of DDR1 monomer, dimer and oligomer, which was normalized to GAPDH and performed using ImageJ based on the analysis of the gray band intensity. n = 4 biological replicates. Data were expressed as the means ± SEM and analyzed by Kruskal-Wallis test with Dunn’s test. P value, ratio of DDR1 dimers and polymers to total DDR1. E Western blotting to assess DDR1 phosphorylation in HUVECs subjected to PS or OS for indicated time. 3 biological replicates were presented. F Schematic diagram of 3D microfluidic vascular model to produce disturbed or laminar flow. G Three-dimensional imaging of DDR1 droplets in HUVECs responding to laminar flow for 2 min. H Left: time-lapse images of HUVECs infected with DDR1-EGFP adenovirus, which seeded on gelatin-coated microfluidic chamber and subjected to laminar flow or disturbed flow for 3 h. DDR1 droplets were indicated by yellow arrows. Right: quantification of DDR1 condensates. n = 6 biological replicates. Data were expressed as the means ± SEM. I Schematic cartoon of aortic arch and thoracic aorta. J Left: En-face staining of DDR1 and VE-cadherin in the aortic arch and thoracic aorta of C57BL/6 wild-type mice. Right: quantification of DDR1 condensates. n = 20 images from 5 mice. K Colocalization analysis of DDR1 and VE-cadherin in ( J ). n = 20 images from 5 mice. Pearson’s R value (above threshold) was calculated by ImageJ Fiji software (Analyze-colocalization-coloc2). In ( J , K ), Data were all expressed as the means ± SEM analyzed by two-sided Mann–Whitney test. Scale bars, 20 μm.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Immunofluorescence, SDS Page, Western Blot, Phospho-proteomics, Imaging, Infection, Staining, Software, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: A Schematic diagram of the magnetic tweezers assay. B, C Images of DDR1 condensates formation (yellow arrows) captured by total internal reflection fluorescence microscopy. HUVECs were infected with DDR1 adenovirus and incubated with collagen/ BSA-coated beads in ( B ), and incubated with anti-DDR1/IgG-coated beads in ( C ). Representative images of HUVECs loaded with Fluo-4AM dye and then incubated with collagen/ BSA-coated beads in ( D ), and anti-DDR1/IgG-coated beads in ( E ). Calcium responses were measured by calculating the fluorescent intensity of individual cells before (10 s), during (120 s), and after (30 s) stimulation. The position of dynabeads is indicated by white dashed circles. n = 16 cells from 4 biological replicates in ( D ). n = 8 cells from 4 biological replicates in ( E ). F Schematic diagram of a membrane-bound tension biosensor (MSS). MSS consists of a tension sensor module, which comprising an elastic spider silk protein inserted between ECFP and YPet, and two anchoring proteins. G The representative living cell images of YPet/ECFP emission ratio and DDR1-Cherry in EA.hy926 ECs subjected to laminar shear stress for 1 min and post-shear for 5 min. DDR1 droplets were indicated by white arrows. H The average time courses of FRET biosensors and quantification of number of DDR1 condensates in EA.hy926 ECs exposed to laminar shear stress. n = 9 biological replicates. I The representative living cell images of YPet/ECFP emission ratio and DDR1-Cherry in EA.hy926 ECs pretreated without or with cholesterol in complex with methyl-β-cyclodextrin (MβCD) (+MβCD-cholesterol). DDR1 droplets were indicated by white arrows. J Left: the average time courses of FRET biosensors in ( I ). Middle: quantification of number of DDR1 condensates in ( I ). Right: quantification of average condensate size in ( I ). n = 9 biological replicates. In ( D , E and J ), Data were all analyzed by two-sided Mann–Whitney test. K Western blotting to assess DDR1 phosphorylation in HUVECs subjected to laminar shear stress for 1 h or static. 3 biological replicates were presented. Data were all expressed as the means ± SEM. In ( B , C ), Scale bars, 5 μm. In ( D , E , G and I ), Scale bars, 10 μm.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Fluorescence, Microscopy, Infection, Incubation, Membrane, Shear, MANN-WHITNEY, Western Blot, Phospho-proteomics

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: A Schematic diagram of single-molecule magnetic tweezers measurements. B The representative force–extension curve of DDR1 extracellular domain protein pretreated without or with shear for 5 min. C Quantification of unfolding force and extension step size of DDR1 extracellular domain protein before- or post-shear. n = 7 biological replicates. Data were analyzed by Wilcoxon matched-pairs signed rank test (two-tailed). D Left: DDR1 extracellular domain crystal structure generated by SWISS-MODEL. Right: the expected structure of DS-like domain-locked mutant (D189C/F364C). E The representative force–extension curve of DDR1 locked mutant (D189C/F364C) pretreated without or with shear for 5 min. F Time-lapse images and quantification of DDR1 condensates in ECs transfected with DDR1 WT or locked mutant. G Live cell imaging and quantification of DDR1 WT or locked mutant in ECs after 10 μg/ml soluble collagen I stimulation. H Time-lapse images and quantification of DDR1 condensates in ECs transfected with DDR1 WT or C287A mutant. I Live cell imaging and quantification of DDR1 WT or locked mutant in ECs after 10 μg/ml soluble collagen I stimulation. In ( F – I ), data were expressed as the means ± SEM and analyzed by two-tailed Mann–Whitney test. n = 8 biological replicates. J, K Left: dual-color FRAP of EA.hy926 ECs expressing DDR1 (WT/locked)-GFP and mCherry-DDR1 (WT/locked). ECs were subjected to 5 min pre-shear before FRET. The photobleaching area was indicated by yellow arrows. Right: schematic of mCherry-DDR1 (WT/locked) and DDR1 (WT/locked)-GFP expressed in ECs treated with anti-mCherry, and quantification of fluorescent intensity of the photobleaching area. n = 24 cells from 6 biological replicates. L Left: non-reducing SDS-PAGE to detect DDR1 oligomerization in EA.hy926 ECs transfected with DDR1-WT or DDR1-D189C/F364C(locked), ECs were subjected to PS for 1 h or static. Right: Quantification of the fraction of DDR1 monomer, dimer and oligomer. n = 4 biological replicates. Data were expressed as the means ± SEM and analyzed by Kruskal-Wallis test with Dunn’s test. P value, ratio of DDR1 dimers and polymers to total DDR1. M Schematic diagram of DS-like domain structure and DDR1 oligomerization before- or post-shear. Data were all expressed as the means ± SEM. In ( F – I ), Scale bars, 10 μm. In ( J , K ), Scale bars, 5 μm.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Shear, Two Tailed Test, Generated, Mutagenesis, Transfection, Live Cell Imaging, MANN-WHITNEY, Expressing, SDS Page

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: A Protein-Protein interaction analysis of the IP-MS identified proteins. B Left: Co-IP between DDR1 and YWHAE in HUVECs cultured under the static condition or subjected to PS or OS for 3 h. IP was performed with anti-DDR1 antibody. HC: Heavy chain. Right: quantification of YWHAE immunoprecipitated with DDR1. n = 4 biological replicates. Data were expressed as the means ± SEM and analyzed by Kruskal–Wallis test with Dunn’s test. C Representative images of NIH3T3 cells expressing Opto-YWHAE. Scale bar, 10 μm. D Left: turbidity of YWHAE solution at different concentrations. Data were expressed as the means ± SD. n = 5 biological replicates. Right: representative fluorescence microscopy images of YWHAE solution at different concentrations. Scale bar, 10 μm. E Representative images of NIH3T3 cells expressing both Opto-YWHAE and DDR1-EGFP. Scale bar, 10 μm. F Schematic representations of full-length DDR1 (DDR1-FL), 416-913aa of DDR1 (DDR1-C) and 21-443aa of DDR1 (DDR1-N). G Cell-free phase separation assay showing droplet formation of YWHAE-Cherry with DDR1 (416-913aa)-EGFP (DDR1-C, 5 μM) or DDR1 (21-443aa)-EGFP (DDR1-N, 5 μM) in indicated concentrations. Scale bar, 10 μm. In ( C , E , and G ), experiments were repeated 3 times independently with similar results. H Turbidity assays of LLPS of DDR1 (416-913aa)-EGFP at different concentrations and YWHAE/DDR1 molar ratio. Data were expressed as the means ± SEM. n = 5 biological replicates. I Up: representative fluorescence microscopy images of DDR1 droplets with different YWHAE concentrations. Down: quantification of fluorescent intensity of the area indicated by the dashed circle after FRAP experiment. n = 6 biological replicates. Data were expressed as the means ± SD. Scale bar, 1 μm. J Representative fluorescence microscopy images of droplets formed by DDR1 with YWHAE in the presence of 1,6-HD or 150 mM NaCl. Scale bar, 10 μm. K Turbidity of DDR1 and DDR1/YWHAE in the presence of 1,6-HD or 150 mM NaCl. n = 6 biological replicates. Data were expressed as the means ± SEM and analyzed by Kruskal–Wallis test with Dunn’s test.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Cell Culture, Immunoprecipitation, Expressing, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: A Live cell imaging of DDR1(1 ~ 669 aa), DDR1(1 ~ 693 aa), DDR1-FL and YWHAE-Cherry in EA.hy926 ECs. ECs were subjected to disturbed flow for 3 h in microfluidic chambers. B Live cell imaging of DDR1-WT/K674A/S677A and YWHAE-WT/Y214A in EA.hy926 ECs. ECs were subjected to disturbed flow for 3 h. C Structural details of the DDR1-YWHAE interfaces generated with PyMol, DDR1 and YWHAE residues are shown as green and red sticks, respectively. D Co-IP between DDR1 and YWHAE in EA.hy926 ECs subjected to PS or OS for 3 h. Cells were transfected with the indicated constructs. IP was performed with anti-DDR1 antibody. Scale bars, 20 μm. In ( A , B , D ), experiments were repeated 3 times independently with similar results.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Live Cell Imaging, Generated, Co-Immunoprecipitation Assay, Transfection, Construct

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: A Immunofluorescence and quantification of YAP localization in HUVECs. The cells were subjected to PS or OS for 24 h. n = 18 images from 3 biological replicates. 10 ~ 15 cells per image. B Western blotting to assess DDR1 and YAP phosphorylation in HUVECs subjected to PS or OS for 24 h. 3 biological replicates were presented. C Quantitative RT-PCR analysis of the expressions of YAP target genes CTGF, CYR61 and ANKRD1 in HUVECs treated as in ( A ). n = 6 biological replicates. D Immunofluorescence and quantification of YAP localization in HUVECs subjected to PS or OS for 24 h. The cells were transfected with siRNAs specific for YWHAE or scrambled siRNA and incubated with DDR1-IN-1 (10 μmol/L) or the control reagent (DMSO) for 3 h before being subjected to shear. n = 18 images from 3 biological replicates. 8 ~ 12 cells per image. E Immunofluorescence and quantification of YAP localization in HUVECs subjected to PS or OS for 24 h. The cells were transfected with si-DDR1, both si-DDR1 and si-YWHAE, or scrambled siRNA. n = 18 images from 3 biological replicates. 12 ~ 16 cells per image. F Western blotting to assess DDR1 and YAP phosphorylation in HUVECs treated as in ( D ). 3 biological replicates were presented. G Quantitative RT-PCR analysis of the expressions of YAP target genes CTGF, CYR61 and ANKRD1 in HUVECs treated as in ( D ). n = 6 biological replicates. Data were all analyzed by two-way ANOVA followed by Tukey’s multiply test. Scale bars, 20 μm. Data were all expressed as the means ± SEM.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Immunofluorescence, Western Blot, Phospho-proteomics, Quantitative RT-PCR, Transfection, Incubation, Control, Shear

Journal: Nature Communications

Article Title: Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation

doi: 10.1038/s41467-023-42341-z

Figure Lengend Snippet: The schematic depicts DDR1 as a primary mechanosensor in ECs, orchestrating cellular responses to shear flow. In areas of atheroprotective laminar flow, YAP phosphorylation prompts its binding with 14-3-3 proteins. This leads to YAP localization in the cytoplasm and subsequent degradation. Conversely, in atheroprone disturbed flow areas, DDR1 perceives the flow, initiating force-induced DDR1 oligomerization. This results in the formation of liquid-like biomolecular condensates involving DDR1 and 14-3-3. These condensates inhibit YAP phosphorylation and cytoplasmic sequestration, resulting in YAP activation and consequent endothelial dysfunction.

Article Snippet: Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting.

Techniques: Shear, Phospho-proteomics, Binding Assay, Activation Assay

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Schematic diagram of wild-type and signalling-defective DDR1 mutants. The extracellular region consists of two globular domains, the N-terminal discoidin (DS) domain and the discoidin-like (DS-like) domain, followed by a highly flexible juxtamembrane (JM) region. The transmembrane (TM) region contains a dimerisation motif. The intracellular catalytic kinase domain is preceded by a large unstructured JM region. The collagen-binding trench in the DS domain is shown in red. Collagen binding to this site in wild-type DDR1 induces phosphorylation of cytoplasmic tyrosine residues in both the JM region and kinase domain (shown as yellow circles). None of the mutants are phosphorylated upon collagen incubation. DDR1-W53A has a mutation in the ligand binding pocket in the DS domain. DDR1-R32E and DDR1-L152E are signalling defective mutants with mutations in the ‘signal patch’ region in the base of the DS domain, near the DS-like domain. DDR1-TM1 is a mutant with impaired transmembrane helix association, and DDR1-K655A is a mutant with impaired catalytic function. The locations of mutations are indicated by red stars, and anti-DDR1 epitopes located in the DS-like domain are symbolised by blue and green ovals for wild-type DDR1.

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Binding Assay, Incubation, Mutagenesis, Ligand Binding Assay

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Collagen I induces DDR1 redistribution on the cell surface. COS-7 cells transiently expressing DDR1 were stimulated with collagen as detailed below. ( A ) Cells were stimulated for the indicated times (in minutes) at 37 °C, then incubated on ice with mAb 7A9 against the DDR1 ectodomain, before fixation and secondary Ab staining. ( B ) Cells were stimulated for the indicated times at 37 °C. Staining was done as above. The graph shows mean ZC scores + SEM (N = 200–400 regions from 80–100 cells from 3 independent experiments). *p < 0.05; ****p < 0.0001 (one-way ANOVA, followed by Bonferroni post hoc test). Data are from a different set of experiments than those shown in panel A. ( C , D ) Cells were stimulated for the indicated times at 37 °C, then fixed and permeabilised, and immunostained for phospho-tyrosine 513 (pY-DDR1) and for DDR1. (C) Mean pY-DDR1 levels across three experiments: mean values were normalised within experiments and then mean values taken for each stimulation time. N is at least 30 for each stimulation time. ( D ) The proportion of cells expressing DDR1 with pY-DDR1 signal above background levels were manually counted for different stimulation times. Mean percentage values ± SEM (N = 55 for all stimulation times, from two independent experiments). ( E ) Cells were either stimulated with collagen I for 10 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9 against the DDR1 ectodomain (shown in green) and anti-collagen-I mAb (shown in magenta), before fixation, and secondary Ab staining. White boxes in left columns indicate corresponding areas shown at higher magnification in columns to the right. All cells were imaged using a widefield microscope. At least 30 cells were imaged for each condition. Scale bars, 30 μm or 10 μm (enlarged images).

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Expressing, Incubation, Staining, Microscopy

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Collagen binding and collagen-induced DDR1 redistribution of signalling defective DDR1 mutants. COS-7 cells transiently expressing WT-DDR1 or the indicated DDR1 mutant were stimulated with collagen I as detailed below. ( A ) Cells were stimulated with collagen for 10 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9, before fixation and secondary Ab staining. White boxes in left columns indicate corresponding areas shown at higher magnification in right columns. ( B ) Cells were stimulated with collagen for 60 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9 (shown in green) and anti-collagen I mAb (shown in magenta), before fixation, and secondary Ab staining. Graph shows the quantified anti-collagen signal with the exclusion of collagen not colocalised with DDR1. The mean collagen-immunostain intensity was calculated for each condition then normalised so that WT-DDR1 collagen-unstimulated and stimulated values were 0 and 100 A.U., respectively. Error bars are SEM (N = 65–121 cells from 3 independent experiments). ( C ) Cells expressing WT-DDR1 were incubated with either collagen I, a mixture of collagen I and anti-DDR1 mAb 7A9, or left unstimulated for 60 minutes at 37 °C. Cells were then immunostained as in B. Arrows indicate collagen aggregates not colocalising with DDR1. Cells were imaged using a widefield microscope. Scale bars, 30 μm or 5 μm (enlarged images in A). For each condition, at least 20 cells were imaged in A, and at least 30 cells were imaged in C.

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Binding Assay, Expressing, Mutagenesis, Incubation, Staining, Microscopy

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Signalling-defective DDR1 mutants bind triple-helical DDR1 selective peptide but do not phosphorylate with peptide stimulation. ( A ) COS-7 cells transiently expressing wild-type DDR1 or the indicated DDR1 mutant were incubated with or without a biotinylated DDR-selective collagen-mimetic peptide for 60 minutes on ice, followed by incubation with anti-DDR1 mAb 7A9 on ice. Cells were then fixed and incubated with Alexa Fluor-488 goat-anti-mouse IgG and Alexa Fluor-546 conjugated streptavidin. Cells were imaged by widefield microscopy. The graph shows mean fluorescence intensity, normalised to respective DDR1 expression levels. N = 27–31 fields of view from 3 independent experiments. Scale bar, 20 μm. ( B ) HEK293 transiently expressing wild-type DDR1 or the indicated DDR1 mutant were stimulated with collagen I (C), or with DDR-selective collagen-mimetic peptide (P) for 60 minutes at 37 °C or were left unstimulated. Cell lysates were analysed by Western blot using an Ab against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). NS, no significance; *p < 0.05; **P < 0.01; ****p < 0.0001 (two-way ANOVA, followed by Tukey’s multiple comparisons test. ( C ) COS-7 cells transiently expressing DDR1 were incubated with collagen-mimetic triple-helical peptide (Peptide) or a control triple-helical peptide without the DDR binding motif (Control) for 0 to 60 minutes at 37 °C, as indicated. Cells were then incubated with anti-DDR1 mAb7A9 Ab on ice, before fixation and incubation with secondary Abs. Lower panels show magnified views of the boxed areas in the upper panels. Cells were imaged by widefield microscopy. Scale bars, 20 μm (upper image) or 10 μm (magnification). Right: ZC score of surface DDR1 staining in cells stimulated with Control or Peptide for 0 to 60 minutes. Data show mean + SEM (N = 200–300 regions from 35–50 cells from 2 independent experiments). NS, no significance; ***p < 0.001 (one-way ANOVA, followed by Bonferroni post hoc test).

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Expressing, Mutagenesis, Incubation, Microscopy, Fluorescence, Western Blot, Binding Assay, Staining

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Anti-DDR1 mAbs block phosphorylation of collagen-bound DDR1. The diagram at the top gives an overview of the experimental procedures. HEK293 cells transiently expressing wide-type DDR1 were first incubated with collagen I for 60 minutes on ice, in the presence (+) or absence (−) of the indicated anti-DDR1 mAbs (Phase 1). Following washes, cells were incubated for 30 minutes at 37 °C, in the absence (no mAb) or presence of the indicated mAbs (Phase 2). The sample shown in lane 1 was lysed immediately after the incubation with collagen on ice. Samples labelled 2 were replicate lysates from 3 different wells. Cell lysates were analysed by Western blot using a mAb against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with rabbit anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). ****p < 0.0001 (one-way ANOVA, followed by Dunnett's multiple comparison test).

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Blocking Assay, Expressing, Incubation, Western Blot

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: SIM images reveal DDR1-immunostain double-walled structures for cells stimulated with collagen for 60 minutes at 37 °C. COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 10 or 60 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9, before fixation and secondary Ab staining. 3D-SIM images were acquired using a Zeiss ELRYA microscope; images are from one of 15 slices. Lower panels show magnified views of the boxed areas in the upper panels. Arrowheads in A indicate example structures wider than 200 nm (full width at half maximum). Scale bars, 30 μm (upper panels) or 5 μm (magnified images). At least 10 cells were imaged for each condition.

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Expressing, Incubation, Staining, Microscopy

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. ( A ) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. ( B , C ) COS-7 cells transiently expressing DDR1-SNAP were incubated with SNAP-Surface Alexa Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes ( B ) or for 5, 10, or 60 minutes ( C ) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining ( B ), or fixed and mounted ( C ). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices ( B ) or from a single slice ( A , C ). White boxes indicate corresponding areas shown at higher magnification in lower images ( B ). Scale bars, 5 μm ( A ), 30 μm (upper image in B), 2 μm (enlarged images in B) or 3 μm ( C ). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal ( B ). At least 10 cells were imaged for each condition.

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Expressing, Incubation, Immunostaining, Staining, Microscopy, Binding Assay

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: mAb-513-induced DDR1 redistribution and phosphorylation. ( A ) COS-7 cells transiently expressing DDR1 were either incubated with mAb-513 IgM for the indicated times at 37 °C or left unstimulated, then incubated on ice with anti-DDR1 mAb 7A9, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Cells were imaged using a widefield microscope. White boxes in DDR1 images indicate corresponding areas shown at higher magnification in images to the right. Scale bars, 30 μm or 10 μm (magnification). ( B ) Cells were treated and imaged as above. Mean pY-DDR1 signal for each cell was calculated and averaged for the stimulation time. Values were normalised so that the mean value for 0 and 60 minutes was 0 and 100 A.U respectively. Error bars are SEM. N = 15–23.

Article Snippet: The following primary Abs were used: rabbit anti-DDR1 (SC-532, Santa Cruz, Dallas, TX; for Western blotting); monoclonal rabbit anti-phospho-DDR1 (Tyr513, E1N8F, Cell Signaling; for Western blotting and immunostaining, validated in ref. ); polyclonal rabbit anti-phospho-DDR1 (Tyr 513, abx012650, Abbexa; for immunostaining); rabbit anti-phospho-DDR1 (Tyr 792, 11994, Cell Signaling, validated in ref. ); rabbit anti-phospho-DDR1/DDR2 (DDR1 Y796, DDR2 Y740, MAB25382, R&D Systems, validated in ref. ); mouse anti-Flag IgG1 clone M2 (Sigma); mouse anti-rat collagen type I (clone 1F10C2, IgG2b; Chondrex).

Techniques: Expressing, Incubation, Immunostaining, Microscopy