Journal: Cellular signalling

Article Title: Vegfr3 activation of Pkd2l1 + CSF-cNs triggers the neural stem cell response in spinal cord injury.

doi: 10.1016/j.cellsig.2025.111675

Figure Lengend Snippet: Fig. 3. Role of Vegfr3 in the proliferation and activation of Pkd2l1+ CSF-cNs. (A, B) The overexpression and knockdown efficiencies of Vegfr3 in Pkd2l1+ CSF-cNs were confirmed through qRT-PCR and Western blotting following transfection. (C) Representative images of CSF-cNs and the average diameter of neurospheres with or without Vegfr3 overexpression or inhibition. (D) Representative immu nofluorescence images and quantification of EdU+ Pkd2l1+ CSF-cNs with or without Vegfr3 overexpression or inhibition. (E) Representative immunofluorescence images and quantification of pHH3+ Pkd2l1+ CSF-cNs with or without Vegfr3 overexpression or inhibition. (F) A CCK-8 assay was used to assess the viability of Pkd2l1+ CSF-cNs with either Vegfr3 overexpression or knockdown. (G) Representative Western blots showing the protein levels of EGFR, Ascl, and Sox2 in Pkd2l1+

Article Snippet: Primary antibodies against various proteins were used as follows: Vegfr1 (#13687–1-AP, 1:500, Proteintech, Wuhan, China), Vegfr2 (#26415–1-AP, 1:1,000, Proteintech, Wuhan, China), Vegfr3 (#20712–1-AP, 1:500, Proteintech, Wuhan, China), Vegfr3 (#ab300403, 1:500, Abcam, Cambridge, UK), GAPDH (#60004–1-lg, 1:5,000, Proteintech, China), EGF receptor (EGFR) (#18986–1-AP, 1:600, Proteintech, China), proliferating cell nuclear antigen (#10205–2-AP, 1:500, Proteintech, China), Sox2 (#11064–1-AP, 1:500, Proteintech, China), Ascl1 (#55467, 1:400, Cell Signaling Technology, MA, United States), β-tubulin (#10094–1-AP, 1:5,000, Proteintech, China), Caspase 3 (#9662, 1:500, Cell Signaling Technology, MA, United States), and Cleaved Caspase 3 (#9661, 1:500, Cell Signaling Technology, MA, United.

Techniques: Activation Assay, Over Expression, Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Inhibition, Immunofluorescence, CCK-8 Assay

Journal: International journal of oncology

Article Title: Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion.

doi: 10.3892/ijo.2011.1002

Figure Lengend Snippet: Figure 1. UVA and UVB induce FAP-α in fibroblasts, melanocytes and primary melanoma cells. Expression of FAP-α in normal human fibroblasts analyzed by immunocytochemistry 4 h after (A) shamtreatment, (B) UVA (6 J/cm2) and (C) UVB (60 mJ/cm2) radiation. Percentage of FAP-α positive cells analyzed by immunocytochemistry (n=4) and protein expression in a representative Western blot in (D) fibroblasts, (E) melanocytes, (F) keratinocytes, (G) primary WM55P, (H) regional metastatic WM55M1 and (I) systemic metastatic WM55M2 melanoma cells 4 h after shamtreatment, UVA and UVB radiation. Horizontal line indicate median of four experiments, *p<0.05.

Article Snippet: The immunodetection was performed by incubating for 2 h at room temperature with the polyclonal primary antibody uPA, Wnt-5a and PDGF-BB (all from santa Cruz Biotechnology), or with one of the following monoclonal primary antibodies: FAP-α, sDF-1α and TGF-β1 (all from santa Cruz Biotechnology).

Techniques: Expressing, Immunocytochemistry, Western Blot

Journal: International journal of oncology

Article Title: Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion.

doi: 10.3892/ijo.2011.1002

Figure Lengend Snippet: Figure 2. FAP-α regulates migration and invasion. Percentage of migrated (A) melanocytes, (B) fibroblasts and (C) primary melanoma cells analyzed by Cultrex® Migration Assay. Percentage of invasive (D, G) melanocytes, (E, H) fibroblasts and (F, I) primary melanoma cells with collagenase I or collagenase IV activity analyzed by Cultrex Collagen I Cell Invasion Assay and Cultrex Collagen IV Cell Invasion Assay. Cells were shamtreated or exposed to UVA (6 J/cm2) or UVB (60 mJ/cm2) prior to assay and then incubated for 24 h. For FAP-α/DPPIV inhibition Gly-ProP(OPh)2 (100 µM, 24 h) was supplemented to the cell culture. (n=4, *p<0.05).

Article Snippet: The immunodetection was performed by incubating for 2 h at room temperature with the polyclonal primary antibody uPA, Wnt-5a and PDGF-BB (all from santa Cruz Biotechnology), or with one of the following monoclonal primary antibodies: FAP-α, sDF-1α and TGF-β1 (all from santa Cruz Biotechnology).

Techniques: Migration, Activity Assay, Invasion Assay, Incubation, Inhibition, Cell Culture

Journal: International journal of oncology

Article Title: Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion.

doi: 10.3892/ijo.2011.1002

Figure Lengend Snippet: Figure 3. Melanocytes and melanoma cells induce FAP-α expression in fibroblasts. Percentage of FAP-α positive fibroblasts analyzed by immunocytochemistry (n=4) and protein expression of the co-cultures in a representative Western blot. Fibroblasts (FB) were either co-cultured with (A) melanocytes (MC), (B) primary WM55P (PM), (C) regional metastatic WM55M1 (M1) and (D) systemic metastatic WM55M2 (M2) melanoma cells or cultured with supernatants of these cells. The cells in (A-D) were shamtreated or exposed to UVA (6 J/cm2) or UVB (60 mJ/cm2) prior to 4 h of co-culture. Horizontal line indicates median of four experiments, *p<0.05.

Article Snippet: The immunodetection was performed by incubating for 2 h at room temperature with the polyclonal primary antibody uPA, Wnt-5a and PDGF-BB (all from santa Cruz Biotechnology), or with one of the following monoclonal primary antibodies: FAP-α, sDF-1α and TGF-β1 (all from santa Cruz Biotechnology).

Techniques: Expressing, Immunocytochemistry, Western Blot, Cell Culture, Co-Culture Assay

Journal: International journal of oncology

Article Title: Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion.

doi: 10.3892/ijo.2011.1002

Figure Lengend Snippet: Figure 4. Supernatant from melanocytes and melanoma cells induce FAP-α driven migration and invasion. Percentage of migrated and invasive fibroblasts analyzed by Cultrex Migration Assay and Cultrex Collagen I Cell Invasion Assay. Fibroblasts (FB) were cultured for 24 h in supernatants derived from shamtreated or UVA (6 J/cm2) or UVB (60 mJ/cm2) irradiated (A, C) melanocytes (MC) and (B, D) primary WM55P (PM) melanoma cells. For FAP-α/ DPPIV inhibition Gly-ProP(OPh)2 (100 µM, 24 h) was supplemented to the cell culture (n=4, *p<0.05).

Article Snippet: The immunodetection was performed by incubating for 2 h at room temperature with the polyclonal primary antibody uPA, Wnt-5a and PDGF-BB (all from santa Cruz Biotechnology), or with one of the following monoclonal primary antibodies: FAP-α, sDF-1α and TGF-β1 (all from santa Cruz Biotechnology).

Techniques: Migration, Invasion Assay, Cell Culture, Derivative Assay, Irradiation, Inhibition

Journal: International journal of oncology

Article Title: Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion.

doi: 10.3892/ijo.2011.1002

Figure Lengend Snippet: Figure 5. PDGF-BB, TGF-β1 and Wnt5a protein induce FAP-α mediated migration and invasion. (A) Effect of supplementation of 10 ng/ml recombinant platelet derived growth factor BB (PDGF-BB), transforming growth factor-β1 (TGF-β1), urokinase-type plasminogen activator (uPA) and Wnt5a on expression of FAP-α in normal human fibroblasts in one representative blot. β-actin served as a positive control. (B) Percentage of migrated fibroblasts analyzed by Cultrex Migration Assay and (C) percentage of invasive fibroblasts analyzed by Cultrex Collagen I Cell Invasion Assay. Fibroblast cell culture medium was supplemented with recombinant PDGF-BB, TGF-β1, uPA, and Wnt5a for 24h. For FAP-α/DPPIV inhibition Gly-ProP(OPh)2 (100 µM, 24 h) was supplemented to the cell culture (n=4, *p<0.05).

Article Snippet: The immunodetection was performed by incubating for 2 h at room temperature with the polyclonal primary antibody uPA, Wnt-5a and PDGF-BB (all from santa Cruz Biotechnology), or with one of the following monoclonal primary antibodies: FAP-α, sDF-1α and TGF-β1 (all from santa Cruz Biotechnology).

Techniques: Migration, Recombinant, Derivative Assay, Expressing, Positive Control, Invasion Assay, Cell Culture, Inhibition

Journal: The FEBS journal

Article Title: Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-α.

doi: 10.1111/j.1742-4658.2011.08051.x

Figure Lengend Snippet: Fig. 1. FAP enzyme activity. (A) Purified soluble recombinant human FAP was incubated with H-Ala-Pro-AMC, H-Gly-Pro-AMC, succinyl-Ala-Pro-AMC and Z-Gly-Pro-AMC fluorescent substrates. (B) Inhibition profile of FAP hydrolysis of Z-Gly-Pro-AMC. Various concentrations of ValboroPro showed dose-dependent inhibition of FAP as compared with buffer alone. Enzyme activity was detected as change in fluorescence units over time.

Article Snippet: This form of DPP4 lacks the cytoplasmic and transmembrane domains, and was purified by immobilized metal affinity chromatography, followed by Superose 12 (GE Healthcare, Uppsala, Sweden), dialysed against 10 mm Tris (pH 8.0), and then stored at 4 C. Purified soluble recombinant human FAP (26–760) was from R&D Systems 1328 FEBS Journal 278 (2011) 1316–1332 a 2011 The Authors Journal compilation a 2011 FEBS (Minneapolis, MN, USA).

Techniques: Activity Assay, Recombinant, Incubation, Inhibition