Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: In vitro citrullination of bacterially produced chemokines leads to their partial degradation. ( a ) Colloidal Coomassie stained SDS-PAGE analysis of the MCP-1/CCL2 products of in vitro citrullination. In vitro citrullination of commercially available recombinant MCP-1/CCL2 and self-made bacterially produced MCP-1/CCL2 performed with recombinant human PAD2 and PAD4 or rabbit PAD2, respectively. Colloidal Coomassie stained MCP-1 bands are shown while recombinant humanPAD2/4 or rabbit PAD2 (Sigma Aldrich/Merck, Waltham, MA, USA) were added to reactions into the quantities below the Colloidal Coomassie sensitivity limits and cannot be visualized. ( b ) Immunoblot analysis of bacterially produced MCP-1/CCL2 upon an in vitro citrullination reaction. Self-made full-length bacterially produced MCP-1/CCL2 was citrullinated in vitro with rabbit PAD2 and resolved on SDS-PAGE, stained with Ponceau S. detection of total MCP-1/CCL2 and modified citrullines with Senshu’s antibody that recognizes that modified citrullines were made according to Senshu’s protocol . Results shown are a representative of three or more repetitive experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: In Vitro, Produced, Staining, SDS Page, Recombinant, Western Blot, Modification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Mass-spectrometry verification of successful in vitro citrullination of MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 (mature form of the protein spans residues 24–99). The first row shows 23 amino acid-long signaling peptide and the second row–mature protein of 76 residues. All arginine residues are highlighted with circles. The boxed areas show the peptide that contains the citrullinated arginine residues that were detected. ( b ) Annotated tandem mass spectrometry (MS/MS) fragmentation spectrum for the citrullinated MCP1/CCL2 peptide, showing the citrullinated arginine residue at position 3 of the studied peptide (R45). The MS/MS fragmentation data were annotated using Expert System (Max Planck Institute of Biochemistry). The precursor ion was observed with a mass error of 1 part per million, and the error for the fragment ions was 0.02 daltons.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Mass Spectrometry, In Vitro, Sequencing, Tandem Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Detection of citrullinated recombinant human MCP-1/CCL2. Standard curves of citrullinated recombinant human MCP-1/CCL2 were set up using enzyme-linked immunosorbent assay in duplicate ( a ) standard curve of citrullinated E. coli produced recombinant human MCP-1/CCL2 chemokine (R&D Systems), ( b ) standard curve for citrullinated MCP-1/CCL2 chemokine produced by and purified from human HEK 293T cells. Absorbance at 450 nm is shown. Coefficients of determination R 2 are indicated in red on the relevant plots. Results shown are a representative of three or more repetitive experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Produced, Purification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Site-directed mutagenesis of potential glycosylation site at asparagine-14 in mammalian cell-produced MCP-1 significantly destabilizes recombinant human MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 with indicated positions of predicted N-glycosylation and citrullination site confirmed by a mass-spectrometry. ( b ) Immunoblot analysis of bacterially versus mammalian cell-produced MCP-1/CCL2 upon an in vitro citrullination reaction. Bacterially produced wild-type MCP-1/CCL2 or HEK293T cell-produced wild-type and N14Q mutant version of chemokine were incubated with EDTA-free proteinase inhibitor cocktail supplemented cellular lysates prepared from the control (vehicle-transfected) or indicated hPAD enzyme-transfected HEK293T cells. Citrullination reactions that were performed directly within cellular lysates essentially as published before . Recombinant chemokine was concentrated with immunoprecipitation and resolved on 10–18% polyacrylamide gradient SDS-PAGE. Results shown are a representative of three experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Mutagenesis, Produced, Recombinant, Sequencing, Mass Spectrometry, Western Blot, In Vitro, Incubation, Transfection, Immunoprecipitation, SDS Page

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Cell Culture, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-α (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-α at the last 24 hours of culture (MSCs + TNF-α); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-α at the last 24 hours of culture (MSCs + MDA CM + TNF-α). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: * P <0.05, ** P ≤0.01, *** P ≤0.001. NS, not significant. # Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Generated, Derivative Assay, Cell Culture, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Basal and cytokine-induced release of inflammatory chemokines by patient CAFs. CAFs were isolated from lung metastasis of a breast cancer patient (CAFs #1) or from a primary tumor of a different breast cancer patient (CAFs #2). (A1, B1) Expression of the pro-malignancy chemokines CCL2, CXCL8 and CCL5 was determined by ELISA, in the linear range of absorbance. (A2, B2) Expression of CCL5 was determined following TNFa and IL-1β stimulation (TNF-α, 50 ng/ml; IL-1β, 500 pg/ml; 48 hours). Control cells were stimulated by vehicle. Expression of the chemokines was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α upregulates the inflammatory profile of MSCs via receptors TNF-RI and TNF-RII. (A) Relative induction of CCL2, CXCL8 and CCL5 in MSCs by TNF-α. Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 24 hours and the expression of the inflammatory chemokines CCL2, CXCL8 and CCL5 in supernatants of MSCs was determined by ELISA, in the linear range of absorbance. Results are from an experiment in which all three chemokines were analyzed in parallel, and the ratios between the three chemokines are representatives of the values obtained in many experimental repeats. (B) Human BM-derived MSCs were exposed to neutralizing antibodies against tumor necrosis factor receptors TNF-RI and TNF-RII or nonrelevant isotype-matched control antibodies (Control Ab) 1 hour prior to stimulation with TNF-α (50 ng/ml), and in the course of 24 hours of stimulation by the cytokine. Control, cells stimulated with vehicle. Expression of CCL2 was determined by ELISA, in the linear range of absorbance. The findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: CCL2 is expressed by CAFs localized in vicinity to breast cancer cells in tumors of invasive ductal carcinoma (IDC) patients. CCL2 expression was determined by immunohistochemistry in biopsy sections of patients diagnosed with IDC: (A) IDC #1, (B) IDC #2. CCL2 staining was detected in cancer cells and in adjacent fibroblasts. Some of the CCL2-expressing CAFs are indicated by arrows.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. # siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Phospho-proteomics, Western Blot, Transfection, Small Interfering RNA, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: NF-κB is essential in mediating TNF-α-induced release of chemokines by MSCs. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 15 minutes. The levels of IκBα (the negative regulator of the NF-κB pathway) were determined by WB analyses. GAPDH was used as a loading control throughout. (B) CAFs were generated by culturing MSCs with Tumor CM from MDA-MB-231 (MDA) or MCF-7 breast tumor cells over a prolonged period of time (~30 days). TNF-α (50 ng/ml) was added for the last 24 hours to MSCs + Tumor CM cells and IκBα levels were determined by WB analyses. (C) CAF #1 cells were stimulated for 48 hours by TNF-α (50 ng/ml). IκBα levels were determined by WB analyses. (D) Human BM-derived MSCs were stimulated with TNF-α (50 ng/ml) for 10 minutes. p65 phosphorylation was determined by WB analyses. (E) Human BM-derived MSCs were transiently transfected by siRNA to p65 or by control siRNA. (E1) p65 expression was determined by WB analyses. (E2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 48 hours. Expression of CCL2 and CXCL8 in the supernatants of the cells was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Generated, Phospho-proteomics, Transfection, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: MSCs promote monocyte migration through TNF-α-induced secretion of CCL2. Supernatants (sups) were collected from human BM-derived MSCs that were stimulated by TNF-α (50 ng/ml) or by its vehicle for 24 hours. Thereafter, the sups were incubated with neutralizing antibodies for CCL2 or with nonrelevant isotype-matched control antibody (I.C.) for 30 minutes. The migration of monocytic cells in response to control medium (without chemokine), to recombinant human CCL2 (rhCCL2; 100 ng/ml) or to sups from TNF-α-stimulated or TNF-α nonstimulated MSCs was determined by Boyden chamber migration assays. Cells were counted under a high-power field (HPF). The findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Migration, Derivative Assay, Incubation, Control, Recombinant

Journal: Cells

Article Title: Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy

doi: 10.3390/cells10010159

Figure Lengend Snippet: Univariate Cox’s regression analysis: Association between chemotherapy, considering the immune markers CCL2, CD68, and CD163, and prognosis stratified by the nodal stage.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with tris-ethylenediaminetetraacetic acid (TE)–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques:

Journal: Cells

Article Title: Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy

doi: 10.3390/cells10010159

Figure Lengend Snippet: Univariate Cox’s regression analysis: Association between chemotherapy, considering the immune markers CCL2, CD68, and CD163, and prognosis stratified by the tumor stage.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with tris-ethylenediaminetetraacetic acid (TE)–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques:

Journal: Cells

Article Title: Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy

doi: 10.3390/cells10010159

Figure Lengend Snippet: Kaplan–Meier analysis: Association of chemotherapy with prognosis (OS, DSS, and RFS) in the N0/N1 + 2 groups stratified by CCL2 expression (>6% or ≤6%). In the N0 group ( A – C ) with CCL2 (>6%), chemotherapy treatment was not associated with OS ( A ) or DSS ( B ) but was associated with shorter RFS ( C ; p = 0.044). In the N1 + 2 group ( D – F ) with CCL2 (≤6%), chemotherapy was not associated with OS ( D ) but was associated with longer DSS ( E ; p = 0.031) and longer RFS (F; p = 0.035).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with tris-ethylenediaminetetraacetic acid (TE)–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cells

Article Title: Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy

doi: 10.3390/cells10010159

Figure Lengend Snippet: Kaplan–Meier analysis: Association of chemotherapy with prognosis (OS, DSS, and RFS) in the pT2/pT3 + 4 groups stratified by CCL2 expression (>6% or ≤6%). In the pT2 group ( A – C ) with CCL2 (>6%), chemotherapy was associated with shorter OS ( A ; p = 0.002), shorter DSS ( B ; p < 0.001), and shorter RFS ( C ; p < 0.001). In the pT3 + 4 group ( D – F ) with CCL2 (≤6%), chemotherapy was not associated with OS ( D ), DSS ( E ), or RFS ( F ).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with tris-ethylenediaminetetraacetic acid (TE)–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cells

Article Title: Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy

doi: 10.3390/cells10010159

Figure Lengend Snippet: Multivariate Cox’s regression analysis: Association between chemotherapy and prognosis stratified by nodal stage and tumor stage (adjusted for the immune markers CCL2, CD68, and CD163).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with tris-ethylenediaminetetraacetic acid (TE)–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques:

Journal: Cells

Article Title: Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy

doi: 10.3390/cells10010159

Figure Lengend Snippet: Multivariate Cox’s regression analysis: Association between immunological markers and prognosis stratified by nodal stage and tumor stage (adjusted for chemotherapy and the immune markers CCL2, CD68, and CD163).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with tris-ethylenediaminetetraacetic acid (TE)–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Marker