Journal: medRxiv

Article Title: Anomalous epithelial variations and ectopic inflammatory response in chronic obstructive pulmonary disease

doi: 10.1101/2020.12.03.20242412

Figure Lengend Snippet: A. Representative images immunostained with CCL2, TNC, and CXCL8 with AT2 cell markers (SFTPC or ABCA3) in COPD and never-smoker lung sections. B . Violin plots for COL4A1, COL4A2, and COL4A3 in AT1 clusters. C. Violin plots for FKBP5 in selected cell types. a) present dataset; b) combined publicly-available datasets.

Article Snippet: Antibodies and reagents were as follows: anti-PD-L1 (rabbit, 1:1000, #ab205921, Abcam, Cambridge, UK); anti-TNC (rabbit, 1:100, #HPA004823, Sigma-Aldrich); anti-CCL2 (rabbit, 1:100, #HPA019163, Sigma-Aldrich); anti-RAGE (rabbit, 1:1000, #ab216329, Abcam); anti-SFTPC (rabbit, 1:1000, #HPA010928, Sigma-Aldrich); anti-ABCA3 (mouse, 1:1000, #WMAB-ABCA3-17, Seven Hills Bioreagents, OH, USA); anti- macrophage inflammatory protein 3 alpha (rabbit, 1:1000, #ab224188, Abcam); anti- CXCL1 (mouse, 1:100, #MAB275, R&D Systems, MN, USA); anti-CXCL8 (mouse, 1:100, #MAB208, R&D Systems); and Hoechst 33242 (1:1000, #H342, Sigma-Aldrich).

Techniques:

Journal: medRxiv

Article Title: Anomalous epithelial variations and ectopic inflammatory response in chronic obstructive pulmonary disease

doi: 10.1101/2020.12.03.20242412

Figure Lengend Snippet: A. 100% stacked bar charts of the percentage of cell populations for AT1 subtype clusters, AT2 subtype clusters, and basal lineage clusters. B. Bar charts displaying the percentages of subpopulations such as AT1-B, AT2-A, AT2-B, club, goblet, and basal clusters across patient states. C. a UMAP plot of epithelial cells focusing on the AT2-C cluster (left). The cell population of the AT2-C cluster (right). The y-axis represents the ratio of cells in the AT2-C cluster across patient states. Brackets () represent the percentage of cells of the AT2-C cluster based on epithelial cells in each of the patient states. D. Violin plots of representative inflammatory-related genes displaying the AT2-C (iAT2) cluster. E. Violin plots for CXCL1 and CXCL8 in AT2 cells in combined publicly-available datasets. F. Representative images immunostained with CD274 (PD-L1), CXCL1 and CXCL20 with AT2 cell markers (SFTPC or ABCA3) in COPD and never-smoker lung sections. Scale bars, 25 μm (left), 12.5 μm (right).

Article Snippet: Antibodies and reagents were as follows: anti-PD-L1 (rabbit, 1:1000, #ab205921, Abcam, Cambridge, UK); anti-TNC (rabbit, 1:100, #HPA004823, Sigma-Aldrich); anti-CCL2 (rabbit, 1:100, #HPA019163, Sigma-Aldrich); anti-RAGE (rabbit, 1:1000, #ab216329, Abcam); anti-SFTPC (rabbit, 1:1000, #HPA010928, Sigma-Aldrich); anti-ABCA3 (mouse, 1:1000, #WMAB-ABCA3-17, Seven Hills Bioreagents, OH, USA); anti- macrophage inflammatory protein 3 alpha (rabbit, 1:1000, #ab224188, Abcam); anti- CXCL1 (mouse, 1:100, #MAB275, R&D Systems, MN, USA); anti-CXCL8 (mouse, 1:100, #MAB208, R&D Systems); and Hoechst 33242 (1:1000, #H342, Sigma-Aldrich).

Techniques:

Journal: bioRxiv

Article Title: Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

doi: 10.1101/2021.11.14.468564

Figure Lengend Snippet: Two colour immunoblot showing cleavage of 150 ng CXCL8 incubated for 2 hours at 37 °C either alone (2 nd lane) or with panel of SpyCEP constructs at a 1:50 molar ratio (SpyCEP:CXCL8). Green bands represent intact CXCL8 (anti-CXCL8 antibody); red bands represent cleaved CXCL8 (anti-ENWVQ). A 17 kDa molecular weight marker is shown in blue, 8 kDa and 6 kDa molecular weights are highlighted by arrows. Figure is representative of 2 independent immunoblots.

Article Snippet: Membranes were subsequently blocked for 1 hour at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques: Western Blot, Incubation, Construct, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

doi: 10.1101/2021.11.14.468564

Figure Lengend Snippet: SpyCEP constructs were co-incubated with CXCL8. Graphs show residual CXCL8 after a 60-minute room temperature incubation, using full length SpyCEP at a 1:1000 ratio to CXCL8; C-terminal at 1:5 – 1:250 molar ratio to CXCL8; and C-terminal S617A mutant at a 1:5 molar ratio to CXCL8. Reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml. N=6 experimental replicates for each construct, data points show means, error bars represent SD. ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, at 60 minutes as determined by ordinary one-way ANOVA.

Article Snippet: Membranes were subsequently blocked for 1 hour at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques: Construct, Incubation, Mutagenesis, Concentration Assay

Journal: bioRxiv

Article Title: Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

doi: 10.1101/2021.11.14.468564

Figure Lengend Snippet: 30-minute, room temperature cleavage time course of: A. 20nM CXCL8 by an equal volume of 50pM SpyCEP and B. 20nM CXCL1 by an equal volume of 100pM SpyCEP. Cleavage reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml and the remaining chemokine measured by ELISA. N=6 experimental replicates for each data point, error bars represent SD of mean values.

Article Snippet: Membranes were subsequently blocked for 1 hour at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

doi: 10.1101/2021.11.14.468564

Figure Lengend Snippet: Data points represent the mean change in cleaved CXCL8 production over time (M s −1 ) of, A. full-length SpyCEP and B. C-terminal SpyCEP). Error bars represent the standard error of the mean of 5 reactions.

Article Snippet: Membranes were subsequently blocked for 1 hour at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 °C with 2 primary antibodies, 1 μg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.

Techniques:

Journal: Oncology Letters

Article Title: Prognostic impact of carbohydrate sulfotransferase 15 in patients with pancreatic ductal adenocarcinoma

doi: 10.3892/ol.2017.6071

Figure Lengend Snippet: Multivariate analysis of disease-free survival.

Article Snippet: Briefly, the sections were incubated with goat anti-human CHST15 antibody (1:200; cat. no. AF3365; R&D Systems, Inc., Minneapolis, MN, USA) for 1 h at room temperature or rabbit anti-human CD44 antibody (1:500; cat. no. ab41478, Abcam, Cambridge, MA, USA) for 1 h at room temperature, and washed with phosphate-buffered solution (PBS).

Techniques:

Journal: Oncology Letters

Article Title: Prognostic impact of carbohydrate sulfotransferase 15 in patients with pancreatic ductal adenocarcinoma

doi: 10.3892/ol.2017.6071

Figure Lengend Snippet: Multivariate analysis of overall survival.

Article Snippet: Briefly, the sections were incubated with goat anti-human CHST15 antibody (1:200; cat. no. AF3365; R&D Systems, Inc., Minneapolis, MN, USA) for 1 h at room temperature or rabbit anti-human CD44 antibody (1:500; cat. no. ab41478, Abcam, Cambridge, MA, USA) for 1 h at room temperature, and washed with phosphate-buffered solution (PBS).

Techniques:

Journal: Oncology Letters

Article Title: Prognostic impact of carbohydrate sulfotransferase 15 in patients with pancreatic ductal adenocarcinoma

doi: 10.3892/ol.2017.6071

Figure Lengend Snippet: CHST15 intensity in PDA tissue. Representative images of PDA tissue stained with CHST15 antibody (upper panel) and H&E (lower panel) are depicted. The negative control was stained using an identical procedure but with the primary antibody omitted. CHST15 staining status was assessed as (A) control, (B) negative, (C) weak, (D) moderate or (E) strong. H&E staining of the same areas of (F) control, (G) negative, (H) weak, (I) moderate and (J) strong tissue. Scale bars, 200 µm; magnification, ×100. CHST15, carbohydrate sulfotransferase 15; PDA, pancreatic ductal adenocarcinoma; H&E, hematoxylin and eosin.

Article Snippet: Briefly, the sections were incubated with goat anti-human CHST15 antibody (1:200; cat. no. AF3365; R&D Systems, Inc., Minneapolis, MN, USA) for 1 h at room temperature or rabbit anti-human CD44 antibody (1:500; cat. no. ab41478, Abcam, Cambridge, MA, USA) for 1 h at room temperature, and washed with phosphate-buffered solution (PBS).

Techniques: Staining, Negative Control, Control

Journal: Oncology Letters

Article Title: Prognostic impact of carbohydrate sulfotransferase 15 in patients with pancreatic ductal adenocarcinoma

doi: 10.3892/ol.2017.6071

Figure Lengend Snippet: Characteristics of patients with strong and negative-to-moderate CHST15 expression.

Article Snippet: Briefly, the sections were incubated with goat anti-human CHST15 antibody (1:200; cat. no. AF3365; R&D Systems, Inc., Minneapolis, MN, USA) for 1 h at room temperature or rabbit anti-human CD44 antibody (1:500; cat. no. ab41478, Abcam, Cambridge, MA, USA) for 1 h at room temperature, and washed with phosphate-buffered solution (PBS).

Techniques: Expressing

Journal: Arthritis Research & Therapy

Article Title: Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

doi: 10.1186/ar1997

Figure Lengend Snippet: CXCL8 and CXCL10 induction in fibroblasts by IL-1β and interferons . Confluent fibroblast monolayers were incubated with IL-1β in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 protein concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Article Snippet: The capture of human CXCL8 in test samples (cell culture supernatants or synovial fluids) was detected by mouse monoclonal anti-human CXCL8 antibody (R&D Systems, Abingdon, UK) and by a secondary antibody, peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Incubation, Protein Concentration

Journal: Arthritis Research & Therapy

Article Title: Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

doi: 10.1186/ar1997

Figure Lengend Snippet: CXCL8 and CXCL10 induction in fibroblasts by tumour necrosis factor alpha and interferons . Confluent fibroblast monolayers were incubated with tumour necrosis factor alpha (TNF-α) in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Article Snippet: The capture of human CXCL8 in test samples (cell culture supernatants or synovial fluids) was detected by mouse monoclonal anti-human CXCL8 antibody (R&D Systems, Abingdon, UK) and by a secondary antibody, peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Incubation, Concentration Assay

Journal: Arthritis Research & Therapy

Article Title: Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

doi: 10.1186/ar1997

Figure Lengend Snippet: CXCL8 and CXCL10 induction in human microvascular endothelial cells by IL-1β and interferons . Human microvascular endothelial cells (HMVEC) were incubated with IL-1β in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Article Snippet: The capture of human CXCL8 in test samples (cell culture supernatants or synovial fluids) was detected by mouse monoclonal anti-human CXCL8 antibody (R&D Systems, Abingdon, UK) and by a secondary antibody, peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Incubation, Concentration Assay

Journal: Arthritis Research & Therapy

Article Title: Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

doi: 10.1186/ar1997

Figure Lengend Snippet: CXCL8 and CXCL10 induction in HMVEC by tumour necrosis factor alpha and interferons . Human microvascular endothelial cells (HMVEC) were incubated with tumour necrosis factor alpha (TNF-α) in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Article Snippet: The capture of human CXCL8 in test samples (cell culture supernatants or synovial fluids) was detected by mouse monoclonal anti-human CXCL8 antibody (R&D Systems, Abingdon, UK) and by a secondary antibody, peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Incubation, Concentration Assay

Journal: Arthritis Research & Therapy

Article Title: Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

doi: 10.1186/ar1997

Figure Lengend Snippet: CXCL8 and CXCL10 in synovial fluid of arthritis patients . (a) CXCL10 and (b) CXCL8 concentrations were measured by ELISAs in synovial fluids of patients with ankylosing spondylitis (AS), psoriatic arthritis (PsA) and rheumatoid arthritis (RA), and were compared with chemokine concentrations in the metabolic arthritis patients with crystal-induced arthritis (CA). The detection limits of the ELISAs for the synovial concentrations of CXCL8 and CXCL10 are indicated on the y axis (logarithmic scale) and were 0.25 ng/ml and 1 ng/ml, respectively. Statistical analysis was performed with the median levels (dashed bars) using the nonparametric Mann–Whitney U test.

Article Snippet: The capture of human CXCL8 in test samples (cell culture supernatants or synovial fluids) was detected by mouse monoclonal anti-human CXCL8 antibody (R&D Systems, Abingdon, UK) and by a secondary antibody, peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: MANN-WHITNEY