Journal: Cell Reports

Article Title: Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability

doi: 10.1016/j.celrep.2021.109237

Figure Lengend Snippet: Stress granules (SGs) inhibit FAO (A and B) Confocal ratiometric imaging of HEK293T cells expressing PercevalHR ATP/ADP ratio marker during starvation for 12 h. Representative confocal planes of the ratio (ex488/ex405) are shown. Scale bar, 5 μm. Graphs show normalized ATP/ADP ratio (ex488/ex405) in starvation and control conditions, mean ± SEM, ∗ p < 0.05. Rotenone (4 μM) was added as a negative control. (C) Oxygen consumption rate (OCR) used for fatty acid β-oxidation (FAO) in HEK293T cells in different conditions. Graph represents FAO dependency in mitochondria to maintain respiration, mean ± SEM, ∗ p < 0.05. (D) Confocal ratiometric imaging of control and HADHA knockout (KO) HEK293T cells expressing PercevalHR during control and starvation. Representative confocal planes are shown. Scale bar, 5 μm. Graph shows average ATP/ADP ratio (ex488/ex405) in starvation and control conditions, mean ± SEM, ∗ p < 0.05. (E) Western blot of HEK293T cells starved for 3 h or treated with PPAR activators clofibrate, rosiglitazone, and GW501516 (100 μM each) for 3 h. Quantification shows fold induction of HADHA relative to control conditions (lane 2), ∗ p < 0.05. (F) Blue graph shows fold change in FAO dependency of HEK293T cells in control medium or starved for 4 or 18 h, and green graph shows ratio of CRISPR-Cas9 PABPC1-DDR2 cells with SGs in the population during 18 h of starvation, mean ± SEM. (G) Confocal microscopy of SG formation in CRISPR-Cas9-tagged PABPC1-DDR2 HEK293T cells incubated in fuel starvation media for indicated amounts of time. Representative confocal planes are shown. Arrows indicate SGs. Scale bar 5, μm. (H) SG formation in cells with or without TDP43 Q331K mutation during starvation. Representative confocal images are shown. Scale bar, 5 μm. Hoechst (10 μg/mL) was used to stain the nucleus. Number in the top left corner of merged image represents percentage of cells with SGs in the population, mean ± SEM, ∗ p < 0.05. (I) FAO dependency in SH-SY5Y cells with or without TDP43 Q331K-GFP mutation during starvation, mean ± SEM, ∗ p < 0.05. (J) Confocal ratiometric imaging of ATP/ADP ratio in SH-SY5Y cells with or without TDP43 Q331K mutation expressing PercevalHR starved for 24 h. Hoechst was added 15 min prior to the imaging to enable cytoplasmic measurement. Representative confocal planes are shown. Scale bar, 5 μm. Graph shows ATP/ADP ratio (ex488/ex405), mean ± SEM, ∗ p < 0.05. (K) Confocal microscopy of oxidative stress levels in SH-SY5Y cells with or without TDP43 Q331K mutation starved for 24 h and stained with 2′,7′-dichlorofluorescin diacetate (H2-DFCDA) (100 μM) 1 h prior to the imaging. Scale bar, 5 μm. (L) Graph shows average cytoplasmic fluorescence intensity in starvation and control conditions, mean ± SEM, ∗ p < 0.01. Top panels: confocal ratiometric microscopy of SHSY-5Y cells with TDP43 Q331K mutation expressing PercevalHR incubated in starvation media for 18 h with vehicle or etomoxir (5 μM). Scale bar, 5 μm. Bottom panels: confocal microscopy of SHSY-5Y cells with TDP43 Q331K mutation incubated in starvation media with vehicle or etomoxir (5 μM) and stained with H2-DFCDA (100 μM) 1 h prior to the imaging. Scale bar, 5 μm. Quantification shows ATP/ADP ratio (ex488/ex405) in starvation and control condition (left) and cytoplasmic fluorescence intensity in starvation and control conditions (right), mean ± SEM, ∗ p < 0.05. (M) Western blot of U2OS WT and G3BP1/2 KO cell lines, ∗ p < 0.01. (N) Confocal microscopy of SGs formation in U2OS WT and G3BP1/2 KO cells grown in control and 16 h starvation conditions, fixed, and stained with anti-G3BP and anti-TIA1 antibodies. Scale bar, 5 μm. Quantification shows the ratio of cells with SGs in the population, mean ± SEM, ∗ p < 0.01. See also A and S3B. (O) Analysis of FAO in U2OS WT and G3BP1/2 KO cells during short-term and long-term starvation, mean ± SEM, ∗ p < 0.01.

Article Snippet: We used the following reagents to detect proteins: monoclonal anti-G3BP (Sigma-Aldrich WH0010146M1), polyclonal anti-TIA1 produced in rabbit (Sigma-Aldrich SAB4301803), anti-GAPDH (sc-47724, Santa Cruz Biotechnology), anti-DDR2 (Origene, clone 1G6), anti-HADHA (sc-374497, Santa Cruz Biotechnology), anti-Tom22 (sc58308, Santa Cruz Biotechnology), anti-VDAC2 (PA528106, Invitrogen), and anti-TARDBP (sc-376532, Santa Cruz Biotechnology), anti-VDAC1 antibody (sc-390996, Santa Cruz Biotechnology).

Techniques: Imaging, Expressing, Marker, Negative Control, Knock-Out, Western Blot, CRISPR, Confocal Microscopy, Incubation, Mutagenesis, Staining, Fluorescence, Microscopy

Journal: Cell Reports

Article Title: Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability

doi: 10.1016/j.celrep.2021.109237

Figure Lengend Snippet: SGs interactome reveals mitochondrial association (A) Confocal microscopy of SG-organelle proximity in HEK293T cells expressing CRISPR-Cas9-tagged PABPC1-DDR2 starved for 9 h. Organelles were visualized with live cell dyes (added 30 min prior to the imaging) or plasmid-encoded markers: LDs (Bodipy-C12), mitochondria (Mito Red), peroxisomes (mCH-SKL), nucleus (Hoechst), lysosome (Lamp1-mCH), endoplasmic reticulum (CALR21-mCH-KDEL), and Golgi apparatus (B4GALT182-mCH). Scale bar, 1 μm. (B) Quantification of SG proximity to membrane-bound organelles showing absolute SG proximity compared with proximity of randomly generated SG sample. Graphs show the ratio of proximal SGs in a sample, mean ± SEM, n = 30. (C) Confocal microscopy of the tripartite association between SG (PABPC1-DDR2), LD (Bodipy-C12), and mitochondria (Rhodamine800) in HEK293T cells expressing CRISPR-Cas9-tagged PABPC1-DDR2 starved for 9 h. Organelles were visualized with live cell dyes (added 30 min prior to the imaging). Kymograph was recorded with 2 s interval between frames for 5 min. Scale bar, 1 μm. (D) Schematic of protein identification by mass spectrometry. (E) Circle diagram shows PABPC1 interactome identified in all conditions. (F) Network of proteins that are present in all SG conditions, but not in the control conditions. Mitochondrial membrane protein is indicated in red. (G) Analysis of SG-enriched fraction by comparison with G3BP-interacting proteins ( Isabelle et al., 2012 ). Mitochondrial membrane protein VDAC2 is indicated in red. (H) Western blot of VDAC1, VDAC2, HADHA, TDP43, and beta-Tubulin in control and starvation conditions. WT HEK293T cells were grown in control and 18 h starvation conditions prior to lysis. Graph shows the ratio of VDAC isoforms to Tubulin in all conditions, mean ± SEM, ∗ p < 0.01. (I) Western blot of VDAC1, VDAC2, G3BP, and beta-Tubulin in control and starvation conditions. WT and G3BP1/2 KO cells were grown in control and 16-h starvation conditions prior to lysis. Graph shows the ratio of VDAC isoforms to Tubulin in all conditions, mean ± SEM, ∗ p < 0.01. (J) Western blot of CRISPR-Cas9 KO of VDAC2, ∗ p < 0.05. (K) GC-FID analysis of FA profiles in purified mitochondria in control and VDAC2 KO cells treated with exogenous FA-CoA. Purified mitochondrial FA profiles were obtained using GC-FID. Graph shows levels of imported exogenous FAs in purified mitochondria, mean ± SEM, ∗ p < 0.05. (L) Analysis of OCR used for FAO in control, VDAC2 KO, and HADHA KO HEK293T cells during short-term starvation, mean ± SEM, ∗ p < 0.05.

Article Snippet: We used the following reagents to detect proteins: monoclonal anti-G3BP (Sigma-Aldrich WH0010146M1), polyclonal anti-TIA1 produced in rabbit (Sigma-Aldrich SAB4301803), anti-GAPDH (sc-47724, Santa Cruz Biotechnology), anti-DDR2 (Origene, clone 1G6), anti-HADHA (sc-374497, Santa Cruz Biotechnology), anti-Tom22 (sc58308, Santa Cruz Biotechnology), anti-VDAC2 (PA528106, Invitrogen), and anti-TARDBP (sc-376532, Santa Cruz Biotechnology), anti-VDAC1 antibody (sc-390996, Santa Cruz Biotechnology).

Techniques: Confocal Microscopy, Expressing, CRISPR, Imaging, Plasmid Preparation, Generated, Mass Spectrometry, Western Blot, Lysis, Purification

Journal: Cell Reports

Article Title: Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability

doi: 10.1016/j.celrep.2021.109237

Figure Lengend Snippet: FA redistribution during SG formation (A and B) HEK293T cells expressing CRISPR-Cas9-tagged PABPC1-DDR2 were starved in a fuel- and serum-depleted media containing FA-free BSA for indicated amounts of time. FA dye (Bodipy-C12, 1 μM) was added 30 min prior to the imaging together with Hoechst (10 μg/mL). Arrows indicate SGs. Scale bar, 5 μm. Graph shows percentage of cells with SGs in the population and LD fluorescence intensity, mean ± SD. Pearson correlation coefficient (r) is 0.98. Fold change in FAO is overlaid. (C and D) Quantification showing increase in accumulation of FAs in LDs during SG formation (SGs were formed by incubation for 60 min with arsenite 100 μM). Graphs show mean ± SEM, n = 30. Intensity profile of FAs accumulation in the LDs. Cells were incubated with arsenite for 1 h. Bodipy (1 μM, green) was added 30 min before the imaging to stain the LDs. FA (Bodipy-C12, 1 μM, red) was added at the start of the acquisition. Confocal images were taken every minute for 15 min. Intensity profiles of the images are shown. Scale bar, 1 μm.

Article Snippet: We used the following reagents to detect proteins: monoclonal anti-G3BP (Sigma-Aldrich WH0010146M1), polyclonal anti-TIA1 produced in rabbit (Sigma-Aldrich SAB4301803), anti-GAPDH (sc-47724, Santa Cruz Biotechnology), anti-DDR2 (Origene, clone 1G6), anti-HADHA (sc-374497, Santa Cruz Biotechnology), anti-Tom22 (sc58308, Santa Cruz Biotechnology), anti-VDAC2 (PA528106, Invitrogen), and anti-TARDBP (sc-376532, Santa Cruz Biotechnology), anti-VDAC1 antibody (sc-390996, Santa Cruz Biotechnology).

Techniques: Expressing, CRISPR, Imaging, Fluorescence, Incubation, Staining

Journal: Cell Reports

Article Title: Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability

doi: 10.1016/j.celrep.2021.109237

Figure Lengend Snippet:

Article Snippet: We used the following reagents to detect proteins: monoclonal anti-G3BP (Sigma-Aldrich WH0010146M1), polyclonal anti-TIA1 produced in rabbit (Sigma-Aldrich SAB4301803), anti-GAPDH (sc-47724, Santa Cruz Biotechnology), anti-DDR2 (Origene, clone 1G6), anti-HADHA (sc-374497, Santa Cruz Biotechnology), anti-Tom22 (sc58308, Santa Cruz Biotechnology), anti-VDAC2 (PA528106, Invitrogen), and anti-TARDBP (sc-376532, Santa Cruz Biotechnology), anti-VDAC1 antibody (sc-390996, Santa Cruz Biotechnology).

Techniques: Recombinant, Clone Assay, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Mesenchymal stem cell induced DDR2 mediates stromal-breast cancer interactions and metastasis growth

doi: 10.1016/j.celrep.2016.12.079

Figure Lengend Snippet: A. DDR2 protein is concordantly expressed in cancer cells and in adjacent mesenchymal stromal cells in human breast cancer metastasis. Immunohistochemical analysis for DDR2 expression in human tissue samples of 21 distant breast cancer metastasis. Shown are representative pictures of metastasis to the bone, brain, small intestine, and liver. Insets show expression of DDR2 in mesenchymal stromal cells (scale bars, 10 µm). Results are tabulated in B.

Article Snippet: Primary antibodies from Cell Signaling included anti-EZH2 (#5246), anti-DDR2 (#12133), anti-DDR1 (#5583), anti-phospho-FAK (Y925) (#3284), anti-FAK (#13009), anti-E-cadherin (#3195), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370), anti-p44/42 MAPK (ERK1/2) (#4695), anti-phospho-SRC Family (Tyr416) (#21010, anti-Src (#2109), anti-SLUG (#9585) and anti-Vimentin (#5741); from Abcam anti-Collagen I (#AB34710) and anti-CK18 (# {"type":"entrez-nucleotide","attrs":{"text":"AB189444","term_id":"51775940","term_text":"AB189444"}} AB189444 ); from R&D Systems phospho-DDR2 (Y740) (#MAB25382); and from Santa Cruz Biotechnology anti-ZEB1 (#SC-81428).

Techniques: Immunohistochemical staining, Expressing

Journal: Cell reports

Article Title: Mesenchymal stem cell induced DDR2 mediates stromal-breast cancer interactions and metastasis growth

doi: 10.1016/j.celrep.2016.12.079

Figure Lengend Snippet: A. Indicated MSCs were lysed and cytosolic (cyto) and nuclear (NE) fractions were isolated for Western blot analyses. The relative distribution of DDR2 and phosphorylated DDR2 (Tyr740) between fractions was determined using densitometry.

Article Snippet: Primary antibodies from Cell Signaling included anti-EZH2 (#5246), anti-DDR2 (#12133), anti-DDR1 (#5583), anti-phospho-FAK (Y925) (#3284), anti-FAK (#13009), anti-E-cadherin (#3195), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370), anti-p44/42 MAPK (ERK1/2) (#4695), anti-phospho-SRC Family (Tyr416) (#21010, anti-Src (#2109), anti-SLUG (#9585) and anti-Vimentin (#5741); from Abcam anti-Collagen I (#AB34710) and anti-CK18 (# {"type":"entrez-nucleotide","attrs":{"text":"AB189444","term_id":"51775940","term_text":"AB189444"}} AB189444 ); from R&D Systems phospho-DDR2 (Y740) (#MAB25382); and from Santa Cruz Biotechnology anti-ZEB1 (#SC-81428).

Techniques: Isolation, Western Blot

Journal: Cell reports

Article Title: Mesenchymal stem cell induced DDR2 mediates stromal-breast cancer interactions and metastasis growth

doi: 10.1016/j.celrep.2016.12.079

Figure Lengend Snippet: A. Immunofluorescence of GFP-231 single cultures, and cocultures of GFP-231+LN-MSC shDDR and GFP-231+LN-MSC shSCR show that DDR2 and nuclear p-DDR2(Tyr740) are upregulated in 231 cells cultured with LN shSCR, and that culture with LN-MSC shDDR2 reverses this effect. The expression of nuclear p-DDR2(Tyr740) was quantified as white nuclear pixels in 50 GFP-231 cells, 4 fields/slide, for each condition, using Image J (scale bars, 25 µm)

Article Snippet: Primary antibodies from Cell Signaling included anti-EZH2 (#5246), anti-DDR2 (#12133), anti-DDR1 (#5583), anti-phospho-FAK (Y925) (#3284), anti-FAK (#13009), anti-E-cadherin (#3195), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370), anti-p44/42 MAPK (ERK1/2) (#4695), anti-phospho-SRC Family (Tyr416) (#21010, anti-Src (#2109), anti-SLUG (#9585) and anti-Vimentin (#5741); from Abcam anti-Collagen I (#AB34710) and anti-CK18 (# {"type":"entrez-nucleotide","attrs":{"text":"AB189444","term_id":"51775940","term_text":"AB189444"}} AB189444 ); from R&D Systems phospho-DDR2 (Y740) (#MAB25382); and from Santa Cruz Biotechnology anti-ZEB1 (#SC-81428).

Techniques: Immunofluorescence, Cell Culture, Expressing

Journal: Cell reports

Article Title: Mesenchymal stem cell induced DDR2 mediates stromal-breast cancer interactions and metastasis growth

doi: 10.1016/j.celrep.2016.12.079

Figure Lengend Snippet: A. Orthotopic tumor formation assay. Representative macroscopic images of mammary tumors formed by GFP-231, GFP-231+DsRED-LN-MSC shSCR, and GFP-231+DsRED-LN-MSC shDDR2 cells injected the mammary fat pads of NOD/SCID mice (n=10 mice per group). Mammary tumors were stained with H&E, pricosirius, and p-DDR2 (scale bars, 50 µm).

Article Snippet: Primary antibodies from Cell Signaling included anti-EZH2 (#5246), anti-DDR2 (#12133), anti-DDR1 (#5583), anti-phospho-FAK (Y925) (#3284), anti-FAK (#13009), anti-E-cadherin (#3195), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370), anti-p44/42 MAPK (ERK1/2) (#4695), anti-phospho-SRC Family (Tyr416) (#21010, anti-Src (#2109), anti-SLUG (#9585) and anti-Vimentin (#5741); from Abcam anti-Collagen I (#AB34710) and anti-CK18 (# {"type":"entrez-nucleotide","attrs":{"text":"AB189444","term_id":"51775940","term_text":"AB189444"}} AB189444 ); from R&D Systems phospho-DDR2 (Y740) (#MAB25382); and from Santa Cruz Biotechnology anti-ZEB1 (#SC-81428).

Techniques: Tube Formation Assay, Injection, 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 ) 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

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: Representative image of DDR2 and collagen type I immunostaining in human breast cancer. ( A – C ): immunostaining of DDR2 in breast cancer cells ( A ), normal breast epithelium ( B ), and human heart as a positive control of DDR2 ( C ). ( D – F ): immunostaining of collagen type I in cancerous stroma ( D ), normal breast stroma ( E ), and human skin as a positive control of collagen type I ( F ). Bar = 100 µm, respectively.

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques: Immunostaining, Positive Control

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: Association between DDR2 status and clinicopathological factors in 224 breast carcinomas.

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques:

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: Association between DDR2 status and clinicopathological factors according to collagen type I status in 224 breast carcinomas.

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques:

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: Association between DDR2 status and clinical outcomes of breast cancer patients (n = 224). ( A – D ): disease-free survival ( A , C ) and breast cancer-specific survival ( B , D ) according to DDR2 status ( A , B ) or DDR2/collagen type I combination status ( C , D ). ( E – H ): disease-free survival according to DDR2 status ( E , F ) or DDR2/collagen type I combination status ( G , H ) in the patients who received chemotherapy ( E , G ) or not ( F , H ).

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques:

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: Uni- and multivariate analysis of disease-free survival.

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques:

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: The effect of DDR2 in the proliferation of human breast cancer cell lines in the presence of collagen type I. ( A ) Immunoblotting of exogenous DDR2 protein in MCF-7, MDA-MB-231, and T47D cells. ( B – D ): cell proliferation of MCF-7 ( A ), MDA-MB-231 ( B ), and T47D ( D ) transfected with an empty vector or DDR2-expressing vector in the absence or presence of collagen coating. ( E ) Immunoblotting of DDR2 in the cells transfected with siRNA against DDR2 (siDDR2-1, 2). ( F – H ): cell proliferation of MCF-7 ( F ), MDA-MB-231 ( G ), and T47D ( H ) transfected with siRNAs in the presence of collagen coating. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the empty vector, respectively.

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: The effect of DDR2 on the resistance to epirubicin in the breast cancer cell lines. ( A – C ): viability of MCF-7 ( A ), MDA-MB-231 ( B ), and T47D ( C ) transfected with an empty vector or DDR2-expressing vector under epirubicin treatment (500 nM for MCF-7; 250 nM for MDA-MB-231 and T47D). These cells were plated onto collagen-coated culture plates. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the empty vector, respectively. ( D – F ): viability of MCF-7 ( D ), MDA-MB-231 ( E ), and T47D ( F ) transfected with siRNA targeting DDR2 under epirubicin treatment. ( G , H ) mRNA and protein expression in chemosensitive parental cells and epirubicin-resistant cells ( G ; MCF-7 series, H ; MDA-MB-231 series). ** p < 0.01, respectively. ( I , J ) The effect of DDR2 inhibitor WRG-28 treatment (48 h) on the proliferation of chemosensitive parental cells and epirubicin-resistant cells ( F ; MCF-7 series, G ; MDA-MB-231 series).

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Cancers

Article Title: Discoidin Domain Receptor 2 Contributes to Breast Cancer Progression and Chemoresistance by Interacting with Collagen Type I

doi: 10.3390/cancers16244285

Figure Lengend Snippet: The effect of DDR2 on the apoptosis of breast MCF-7 ( A ), MDA-MB-231 ( B ), and T47D ( C ). *** p < 0.001.

Article Snippet: The rabbit polyclonal antibody against DDR2 was purchased from GeneTex (Irvine, CA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Serpine1 Regulates the Enhanced Inhibitory Effect of CHIR99021 Combined with Fibroblast Growth Factor 2 on Myocardial Fibrosis After Myocardial Infarction in Mice

doi: 10.3390/ijms27041627

Figure Lengend Snippet: Identification of cardiac fibroblasts and the effect of CHIR99021 and FGF2 on cell viability. ( A ) Immunofluorescence detection of Vimentin and DDR2 expression in purified CFs. Scale bar: 100 μm. ( B ) Quantitative analysis of Vimentin- and DDR2- positive cells. ( C ) Effect of CHIR99021 at different concentrations on CF viability, assessed by CCK-8 assay. ( D ) Effect of CHIR99021 and FGF2 on CF viability, evaluated using Calcein AM staining. Scale bar: 100 μm. ( E ) Quantification of Calcein AM fluorescence intensity. n = 3. * p < 0.05, *** p < 0.001.

Article Snippet: CF-specific markers Vimentin (ab92547, Abcam, 1:250, Shanghai, China) and DDR2 (A01698-1, Boster, 1:200, Wuhan, China) were detected by immunofluorescence staining.

Techniques: Immunofluorescence, Expressing, Purification, CCK-8 Assay, Staining, Fluorescence