mouse anti pdgfra  (R&D Systems)

Journal: Cell death & disease

Article Title: Atypical chemokine receptor ACKR3/CXCR7 controls postnatal vasculogenesis and arterial specification by mesenchymal stem cells via Notch signaling.

doi: 10.1038/s41419-020-2512-2

Figure Lengend Snippet: Fig. 2 PDGFRα and PDGFRβ are essential for VEGF-induced CXCR7 expression in MSCs. The mRNA levels (a), protein levels (b), and relative protein densities (c) of CXCR7 in ihMSCs incubated with indicated concentrations of VEGF for 18 h. *p < 0.05 compared with the control (untreated) group. The CXCR7 mRNA levels (d) and its cell surface levels (e) in ihMSCs pretreated with the neutralizing anti-PDGFRα or anti-PDGFRβ antibody followed by stimulation with VEGF (12 ng/ml) for 24 h. Data are means ± SD (n = 9). *p < 0.001 compared with the control (untreated) group. #p < 0.01 compared with IgG-treated groups. f, g Verification of PDGFRα and PDGFRβ knockdown by siRNAs. Data are means ± SD (n = 6). *p < 0.001 compared with scramble (Scr.) siRNA. The CXCR7 mRNA levels (h) and its cell surface levels (i) in ihMSCs transfected with or without scramble (Scr.), PDGFRα or PDGFRβ siRNAs for 24 h followed by stimulation with VEGF (12 ng/ml) for 24 h. Data are means ± SD (n = 9). *p < 0.0001 compared to the control (untreated) group. #p < 0.01 compared with scramble (Scr.) siRNA.

Article Snippet: The following antibodies were used: β-actin (A5316, Sigma-Aldrich, 1:10,000 dilution), CXCR7 (GTX100027, GeneTex Inc., 1:500 dilution), PDGFRα (MAB322, R&D Systems, 1:2000 dilution), PDGFRβ (MAB1263, R&D Systems, 1:1000 dilution), VEGFR1 (AF321, R&D Systems, 1:1000 dilution), VEGFR2 (AF357, R&D Systems, 1:1000 dilution), VEGFR3 (AF349, R&D Systems, 1:1000 dilution), PLC-γ1 (#2822, Cell Signaling, 1:1000 dilution), phospho-PLC-γ1 (#2821, Cell Signaling, 1:1500 dilution), MEK-1/2 (#9122, Cell Signaling, 1:1000 dilution), phospho-MEK-1/2 (#9121, Cell Signaling, 1:1500 dilution), Akt (#9272, Cell Signaling, 1:1000 dilution), phospho-Akt (#9271, Cell Signaling, 1:1500 dilution), NOTCH1 (ab52627, Abcam, 1:1000 dilution), JAG1 (ab7771, Abcam, 1:1000 dilution), JAG2 (ab226814, Abcam, 1:1000 dilution), DDL4 (MAB1389, R&D Systems, 1:1000 dilution), HEY1 (GTX118007, GeneTex Inc., 1:1000 dilution), EPHB2 (AF467, R&D Systems, 1:1000 dilution) and NRP1 (AF3870,R&D Systems, 1:500).

Techniques: Expressing, Incubation, Control, Knockdown, Transfection

Journal: Cell death & disease

Article Title: Atypical chemokine receptor ACKR3/CXCR7 controls postnatal vasculogenesis and arterial specification by mesenchymal stem cells via Notch signaling.

doi: 10.1038/s41419-020-2512-2

Figure Lengend Snippet: Fig. 3 PDGFR-mediated PI3K signaling is required for VEGF or PDGF-induced CXCR7 expression. The CXCR7 mRNA levels (a) and its cell surface levels (b) in ihMSCs pretreated with vehicle (DMSO), U-73122 (phospholipase C inhibitor, 10 μM), U0126 (MEK inhibitor, 20 μM), or LY294002 (PI3K inhibitor, 10 μM) for 30 min followed by stimulation with VEGF (12 ng/ml) for 24 h. Data are means ± SD (n = 9). *p < 0.01 compared with the control (untreated) group. The CXCR7 mRNA levels (c) and its cell surface levels (d) in ihMSCs pretreated for 30 min with vehicle (DMSO) or LY294002 (10 μM) followed by stimulation with PDGF-BB (10 ng/ml) for 24 h. e The percentage of CD31+ endothelial cells for ihMSCs treated with or without LY294002 followed by stimulation with PDGF-BB for 7 days. Data are means ± SD (n = 9). *p < 0.01 compared with the control (untreated) group. #p < 0.0001 compared with the vehicle group.

Article Snippet: The following antibodies were used: β-actin (A5316, Sigma-Aldrich, 1:10,000 dilution), CXCR7 (GTX100027, GeneTex Inc., 1:500 dilution), PDGFRα (MAB322, R&D Systems, 1:2000 dilution), PDGFRβ (MAB1263, R&D Systems, 1:1000 dilution), VEGFR1 (AF321, R&D Systems, 1:1000 dilution), VEGFR2 (AF357, R&D Systems, 1:1000 dilution), VEGFR3 (AF349, R&D Systems, 1:1000 dilution), PLC-γ1 (#2822, Cell Signaling, 1:1000 dilution), phospho-PLC-γ1 (#2821, Cell Signaling, 1:1500 dilution), MEK-1/2 (#9122, Cell Signaling, 1:1000 dilution), phospho-MEK-1/2 (#9121, Cell Signaling, 1:1500 dilution), Akt (#9272, Cell Signaling, 1:1000 dilution), phospho-Akt (#9271, Cell Signaling, 1:1500 dilution), NOTCH1 (ab52627, Abcam, 1:1000 dilution), JAG1 (ab7771, Abcam, 1:1000 dilution), JAG2 (ab226814, Abcam, 1:1000 dilution), DDL4 (MAB1389, R&D Systems, 1:1000 dilution), HEY1 (GTX118007, GeneTex Inc., 1:1000 dilution), EPHB2 (AF467, R&D Systems, 1:1000 dilution) and NRP1 (AF3870,R&D Systems, 1:500).

Techniques: Expressing, Control

Journal: Molecular Cancer Research

Article Title: Crenolanib Regulates ERK and AKT/mTOR Signaling Pathways in RAS/BRAF-Mutated Colorectal Cancer Cells and Organoids

doi: 10.1158/1541-7786.mcr-20-0600

Figure Lengend Snippet: Figure 1. Gene mutation and expression analysis of crenolanib targeting molecules in clinical tumors. A, Manhattan plots of mutated genes from the TCGA database. Genes with more than 3% mutation are depicted on their chromosomal location. Names of genes with a high mutation rate are shown. B, Enriched pathways in AML and colorectal cancer. Pathways that were significantly enriched (P<0.05) are shown in AML. For colorectal cancer, only the top10 significantly enriched pathways are shown. C, mRNA expressions and mutations of crenolanib-targeting tyrosine kinase receptors. Gene expression levels in individual cases are depicted, and information on gene mutations are indicated by color. D, Immunohistochemical staining of PDGFRA, PDGFRB, and CK20 in colorectal cancer tissues; scale bars, 100 mm. E, A dimensionality reduction step using t-SNE. The analysis of t-SNE revealed a representative population that consists of colorectal cancer tissues, such as cancer cells, stromal cells, and white blood cells. F, The extracted cancer cells were shown. Cells expressing PDGFRA and PDGFRB are shownwithwarm or cold colors, corresponding to high or low expressions, respectively (N ¼ 5,000).

Article Snippet: After deparaffinization and blocking, sections were incubated with primary anti-CK20 rabbit monoclonal antibody (1:800, CST 13063, Cell Signaling Technology) and anti-PDGFRA mouse monoclonal antibody (25 mg/mL, MAB322–500, R&D systems) or anti-PDGFRB mouse monoclonal antibody (1:50, 610113, BD Transduction Laboratories) overnight at 4 C. Thereafter, the sections were probed with a secondary antibody (MACH 2 double stain polymer detection kit #2, Biocare Medical) for 30 minutes at ambient temperature.

Techniques: Mutagenesis, Expressing, Gene Expression, Immunohistochemical staining, Staining

Journal: Molecular Cancer Research

Article Title: Crenolanib Regulates ERK and AKT/mTOR Signaling Pathways in RAS/BRAF-Mutated Colorectal Cancer Cells and Organoids

doi: 10.1158/1541-7786.mcr-20-0600

Figure Lengend Snippet: Figure 2. Effects of crenolanib on colorectal cancer cell lines. A, Relative expression levels of PDGFRA and PDGFRB in four colorectal cancer cell lines; (N ¼ 3). B, The curves of cell viability of four colorectal cancer cell lines by the concentration gradient of crenolanib are shown; (N ¼ 4). C, Representative figures of DLD1 and proliferation curves of HCT116 and DLD1 in vivo treated with crenolanib and without crenolanib (control; N ¼ 5). D, Representative concentration–effect relationships illustrating the effects of combinations of anticancer drugs and crenolanib (0 and 1 mmol/L) in DLD1 cells (N ¼ 4). E, IC50 values of anticancer drugs in DLD1 cells with crenolanib (1 mmol/L) and without crenolanib (0 mmol/L). F, Proliferation curves of HCT116 in vivo treated with oxaliplatin and with/without crenolanib; (N ¼ 5). Data are presented as the mean standard error of the mean. , P < 0.05.

Article Snippet: After deparaffinization and blocking, sections were incubated with primary anti-CK20 rabbit monoclonal antibody (1:800, CST 13063, Cell Signaling Technology) and anti-PDGFRA mouse monoclonal antibody (25 mg/mL, MAB322–500, R&D systems) or anti-PDGFRB mouse monoclonal antibody (1:50, 610113, BD Transduction Laboratories) overnight at 4 C. Thereafter, the sections were probed with a secondary antibody (MACH 2 double stain polymer detection kit #2, Biocare Medical) for 30 minutes at ambient temperature.

Techniques: Expressing, Concentration Assay, In Vivo, Control

Journal: Molecular Cancer Research

Article Title: Crenolanib Regulates ERK and AKT/mTOR Signaling Pathways in RAS/BRAF-Mutated Colorectal Cancer Cells and Organoids

doi: 10.1158/1541-7786.mcr-20-0600

Figure Lengend Snippet: Figure 4. Effects of crenolanib on patient-derived organoids (PDO). A, Representative histological examinations of xenograft tumors from PDOs. Pathological diagnosis of the parental tumor is indicated in parentheses; scale bars, 100 mm. B, Principal component analysis of six colorectal cancer cell lines (red), three normal colonic mucosal tissues (blue), and 11 PDOs (gray). C, PDGFRA and PDGFRB mRNA expression in nine PDOs. Data are normalized against the expression level of the GAPDH gene; (N ¼ 3). D, The curves of cell viability of nine colorectal cancer PDOs by the concentration gradient of crenolanib are shown; (N ¼ 4). E, Relationships between crenolanib IC50 values and PDGFRAor PDGFRB mRNA expression levels in PDOs are shown. F, Cell survival rates of nine PDOs treated with cetuximab (100 nmol/L) and crenolanib (4 mmol/L) are shown; (N ¼ 4). Data are presented as the mean standard error of the mean.

Article Snippet: After deparaffinization and blocking, sections were incubated with primary anti-CK20 rabbit monoclonal antibody (1:800, CST 13063, Cell Signaling Technology) and anti-PDGFRA mouse monoclonal antibody (25 mg/mL, MAB322–500, R&D systems) or anti-PDGFRB mouse monoclonal antibody (1:50, 610113, BD Transduction Laboratories) overnight at 4 C. Thereafter, the sections were probed with a secondary antibody (MACH 2 double stain polymer detection kit #2, Biocare Medical) for 30 minutes at ambient temperature.

Techniques: Derivative Assay, Biomarker Discovery, Expressing, Concentration Assay