Journal: Nucleic Acids Research

Article Title: Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ

doi: 10.1093/nar/gkab980

Figure Lengend Snippet: Phospho-Ku70 causes rapid dissociation from sites of DNA damage; inhibition of neddylation compromises both pKu70 release and DNA repair following laser micro-irradiation. ( A ) Ku70 release from DNA damage sites after the inhibition of neddylation by MLN4924 (3 μM, 1 h), followed by laser micro-irradiation as in Figure . Relative fluorescence intensity fold change (established as in Figure ). Unpaired t-test, NS = non-significant. ( B ) Western blot of γ-H2AX after irradiation alone (a) or after combined cell treatment (b) with MLN4924 (3 μM, 1 h), followed by irradiation (4Gy). Anti-Ku70 (clone N3H10) was used as the loading control. ( C , a) Western blot analyses of the neddylation patterns of untreated, irradiated (4 Gy), or MLN4924 (3 μM, 1 h) pre-treated and irradiated (4Gy) cells expressing eGFP-ser- or -ala-Ku70-fusion proteins. Cells were lysed 2 h post-irradiation. After SDS-PAGE, the membranes were probed with anti-NEDD8 or anti-Ku70 antibodies. The band below the 100 KDa-MW marker with decreased intensity upon MLN4924 treatment indicates decreased neddylation of cullin. (b) The ubiquitylation pattern analysed in protein extracts from cells expressing eGFP-Ku70-fusion proteins. Cells expressing eGFP-ser-Ku70 or eGFP-ala-Ku70 were irradiated with or without MLN4924 or TZ9 pre-treatment and lysed 2 h post-irradiation. Protein extracts were immunoprecitpitated by using a GFP-Trap_MA kit (Chromotek). After SDS-PAGE, the membranes were probed with an anti-ubiquitin, anti-Ku70, or anti-GFP antibody.

Article Snippet: eGFP-ser-Ku70 and eGFP-ala-Ku70 fusion proteins were immunoprecipitated using the ChromoTek GFP-Trap kit according to the supplier's protocol, except for the cell lysis step.

Techniques: Inhibition, Irradiation, Fluorescence, Western Blot, Expressing, SDS Page, Marker

Journal: Nucleic Acids Research

Article Title: Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ

doi: 10.1093/nar/gkab980

Figure Lengend Snippet: Ser-Ku70 stimulates HR activity while ala-Ku70 promotes DNA DSB distal end junctions. ( A ) HR activity and the cell cycle were assessed by flow cytometry of U2OS cell line containing the integrated reporter construct pDR-GFP (a and b). This cell line was transfected using ser-, ala-, or glu-Ku70 vectors as described in the methods section. (c) pKu70 expression was verified by western blot and is shown as cropped blots. The reporter is composed of two inactive eGFP genes. The upstream GFP gene is truncated; the gene downstream of the promoter has an integrated I-SceI cleavage site and is therefore inactive. Upon I-SceI expression, as verified by western blotting (c) (α-tubulin was used as the loading control), the cleaved GFP gene recombines with the truncated GFP gene on the sister chromatid, resulting in the expression of GFP, which was measured by flow cytometry 72h after I-SceI transfection (exemplified in a). The results (d) represent values that were calculated as follows: (I-SceI – transfection events) – (control – transfection events). n = 6. Unpaired t -test, * P < 0.01. ( B ) NHEJ activity was measured (a) using the human fibroblast cell line GC92 (SV40-transformed) expressing ser-, ala-, or glu-Ku70 [controlled by western cropped (b) blots] that contains the intrachromosomally integrated pCOH-CD4 (cohesive ends) reporter construct. Upon cleavage by I-SceI, a fragment containing the H2Kd and CD8 genes was excised. Re-joining of the flanking ends by NHEJ brought the pCMV promoter closer to the CD4 gene, which promoted its expression. (a) CD4 expression on the cell surface was quantified by flow cytometry 72 h after I-SceI transfection. (c) The results represent values that were calculated as follows: (I-SceI – transfection events) – (control – transfection events). n = 4. Unpaired t -test, * P < 0.01. Differences between values of cells expressing ser-Ku70 or glu-Ku70 are statisticaly non significant.

Article Snippet: eGFP-ser-Ku70 and eGFP-ala-Ku70 fusion proteins were immunoprecipitated using the ChromoTek GFP-Trap kit according to the supplier's protocol, except for the cell lysis step.

Techniques: Activity Assay, Flow Cytometry, Construct, Transfection, Expressing, Western Blot, Transformation 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