rat monoclonal anti cxcl1 igg  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Breaking the symmetry of cell contractility drives tubulogenesis via CXCL1 polarization.

doi: 10.1073/pnas.2315894121

Figure Lengend Snippet: Fig. 3. Soft matrix induces CXCL1 polarization promoting cell protrusions and tubulogenesis. Analysis of (A) Cxcl1 mRNA expression of cells grown on the fish skin–derived collagen gel–coating or gel for 6 h and their (B) CXCL1 protein level in the culture supernatant. **P < 0.01; ***P < 0.001. (C) Representative confocal microscopy images showing CXCL1 (green), pT18S19MLC2 (red), microtubules (cyan), and nucleus (blue) in cells on the fish skin–derived collagen gel or gel-coating for 6 h. (D) Statistical analysis of mean fluorescence intensity of CXCL1 per high power field (HPF) in C. There were approximately 61 and 104 cells per HPF in the gel-coating and gel group, respectively. ***P < 0.001. Analysis of (E) Cxcl1 mRNA expression of cells grown on the 9-mo-old rat tail collagen gel with or without sonication (75% amplitude) for 1 d and their (F) CXCL1 protein level in the culture supernatant. ***P < 0.001. (G) Representative epifluorescent microscopy images of cellular staining for microtubules (green), microfilaments (red), and nucleus (blue) in cells treated with SB225002 on the fish skin–derived collagen gel for 7 h (yellow arrows: cell protrusions). (H) Montage images extracted from timelapse recordings of cells treated with siRNAs on fish skin–derived collagen gel (Movie S4) (red arrows; cell protrusions at 6 h). (I) Statistical analysis of relative cell protrusion activity per high power field (HPF) in Fig. 4H. There were approximately 310, 267, and 247 cells per HPF at 1 h in the siCntl, siCxcl1, and siCxcr1 group, respectively. **P < 0.01. (J) Tube formation on fish skin–derived collagen gel for 2 d in cells with siRNA knockdown of CXCL1. Cells with siCntl served as control. ***P < 0.001. (K) Tube formation on fish skin–derived collagen gel for 1 d in cells treated with SB225002 CXCRs inhibitor. *P < 0.05; **P < 0.01. (L) Tube formation on the fish skin–derived collagen gel for 2 d in cells with siRNA knockdown of CXCR1. ***P < 0.001.

Article Snippet: Immunofluorescent staining was performed using specific antibodies against CXCL1 (AF- 515- NA, R&D Systems), tubulin- α (05- 829, Merck), pMLC2 (95777, Cell Signaling Technology), and E- cadherin (610182, BD Biosciences) followed by Alexa Fluor- conjugated secondary antibodies (Invitrogen).

Techniques: Expressing, Derivative Assay, Confocal Microscopy, Fluorescence, Sonication, Microscopy, Staining, Activity Assay, Knockdown, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Breaking the symmetry of cell contractility drives tubulogenesis via CXCL1 polarization.

doi: 10.1073/pnas.2315894121

Figure Lengend Snippet: Fig. 4. Reduction of cell contractility promotes cell protrusions. (A) Real-time PCR analysis of Cxcl1 expression in cells treated with or without blebbistatin on fish skin–derived collagen gel for 1 d. (B) ELISA of CXCL1 protein expression in the cell culture supernatant from cells treated with or without blebbistatin on fish skin–derived collagen gel for 1 d. (C) Representative confocal microscopy images showing CXCL1 (green), microtubules (red), microfilaments (cyan), and nucleus (blue) in cells treated with or without blebbistatin and grown on fish skin–derived collagen gel for 6 h. (D) Representative epifluorescent microscopy images of CXCL1 (green), microtubules (red), and nucleus (blue) in cells treated with or without blebbistatin on fish skin–derived collagen gel for 6 h. Cell morphology was observed through the bright field channel (Ph) (yellow arrows: cell protrusions). (E) Statistical analysis of cell protrusions per high-power field (HPF) of D. ***P < 0.001.

Article Snippet: Immunofluorescent staining was performed using specific antibodies against CXCL1 (AF- 515- NA, R&D Systems), tubulin- α (05- 829, Merck), pMLC2 (95777, Cell Signaling Technology), and E- cadherin (610182, BD Biosciences) followed by Alexa Fluor- conjugated secondary antibodies (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Confocal Microscopy, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Breaking the symmetry of cell contractility drives tubulogenesis via CXCL1 polarization.

doi: 10.1073/pnas.2315894121

Figure Lengend Snippet: Fig. 6. Suppressing the mechanosensing machinery CD29 abolishes CXCL1 expression and tubulogenesis. (A) Western blot analysis showing the expression of CD29 and tubulin-α in cells transfected with control (shCntl) or CD29-targeting shRNA (shCD29). (B) Tube formation on fish skin–derived collagen gel for 1 d in cells with shCntl or shCD29. ***P < 0.001. (C) Representative confocal microscopy images of cells with phalloidin (red) for microfilaments and DAPI (blue) for the nucleus on FITC-conjugated collagen gel for 1 d. (D) Analysis of Cxcl1 mRNA expression in cells grown on fish skin–derived collagen gel for 1 d. ***P < 0.001. (E) ELISA analysis of cell culture supernatant taken from cells grown on fish skin–derived collagen gel for 1 d. ***P < 0.001. (F) Flow chart shows breaking the symmetry of cell contractility by reducing matrix stiffness or myosin activity promotes CXCL1 polarization at cell protrusions. CXCL1–CXCR1 interaction at cell protrusions promotes cell polarization and ECM remodeling, which in turn, accelerates cell–cell connection and tube elongation.

Article Snippet: Immunofluorescent staining was performed using specific antibodies against CXCL1 (AF- 515- NA, R&D Systems), tubulin- α (05- 829, Merck), pMLC2 (95777, Cell Signaling Technology), and E- cadherin (610182, BD Biosciences) followed by Alexa Fluor- conjugated secondary antibodies (Invitrogen).

Techniques: Expressing, Western Blot, Transfection, Control, shRNA, Derivative Assay, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay

Journal: Mucosal immunology

Article Title: Enteric glial adenosine 2B receptor signaling mediates persistent epithelial barrier dysfunction following acute DSS colitis.

doi: 10.1038/s41385-022-00550-7

Figure Lengend Snippet: Fig. 5 Glial A2BRs affect immune mediators in the intestine. a, b Multiplex assay of colon homogenates from mice after the peak (P-DSS) and resolution of acute intestinal inflammation (R-DSS). a A heatmap showing changes in protein expression relative to healthy controls at P-DSS (left) and R-DSS (right). Shades of blue, white, and red depict decreased, unchanged, and increased protein abundance, respectively. Asterisks mark glial A2BR-dependent process as shown in (b). b Protein concentration of CXCL1 and IL-17 at P-DSS (left) and R-DSS (right). *P < 0.045, one-sided 2-way ANOVA, Sidak’s multiple comparisons test. N = 3–6 mice per group. c–g Flow cytometry of cells dissociated from colon lamina propria from mice at the resolution of the DSS-colitis (R-DSS). c Gating strategy for selection of single live cells using forward and side scatter (FSC and SSC). d Gating for CD45+, Ly-6G+, CD3+, and IL-17+ cells were adjusted using fluorescence minus one controls. e–g Summary and analysis of cell populations such as CD45+ (e), double positive CD45+ and Ly-6G+, CD3+, or IL-17+ (f), and double postitive IL-17+ and Ly-6G+ or CD3+ (g). **P = 0.004, 2-way ANOVA, Sidak’s multiple comparisons test. N = 4–6 mice per group.

Article Snippet: One day after the completion of the DSS treatment, mice received an intraperitoneal injection of 100 μg of either a rat anti-mouse CXCL1 (R and D Systems Cat# MAB453, RRID: AB_2087696) or a rat IgG2A isotype control (R and D Systems Cat# MAB006, RRID: AB_357349).

Techniques: Multiplex Assay, Expressing, Quantitative Proteomics, Protein Concentration, Flow Cytometry, Selection

Journal: Mucosal immunology

Article Title: Enteric glial adenosine 2B receptor signaling mediates persistent epithelial barrier dysfunction following acute DSS colitis.

doi: 10.1038/s41385-022-00550-7

Figure Lengend Snippet: Fig. 6 Glial A2BR signaling controls the glial production of the key immune mediators. a Expression of immune mediators whose whole tissue protein expression was affected by the glial A2BR signaling (Fig. 5a) obtained using a previously published dataset of mouse colon in situ glial translating mRNA transcriptome40. Data are shown as median ± range. Selected immune mediators that were expressed by enteric glia in saline- and 2,4-dinitrobenzene sulfonic acid (DNBS)-treated mice were further tested on primary glia (b). N = 3 mice per group. b Proinflammatory cytokine IL-1b stimulates the glial production of key immune signaling molecules. Primary myenteric glia were incubated with vehicle or IL-1β. Expression of selected mediators was determined by qPCR and normalized to the vehicle controls. N = 6 mice per group. c In vivo neutralization with anti-CXCL1 antibody. N = 4–7 mice per group. d, e A2BR signaling has diverse and distinct effects on the production of immune mediators in primary enteric glial cells. Cultured cells were stimulated with selective adenosine receptor agonist NECA (d) or proinflammatory mediator IL-1β (e) alone or in the presence of the selective A2BR inhibitor PSB 603. d A2BR signaling suppresses basal expression of Cxcl1 and Cxcl10 in cultured enteric glia. *P < 0.013, **P < 0.005; ***P = 0.0009, 2-way ANOVA, Sidak’s multiple comparisons test. N = 6–8 mice. e IL-1β-induced expression of glial Il-6 partly relies on A2BR signaling while A2BR inhibition has additive effects on IL-1β-induced expression of Csf3, Cxcl1, and Cxcl10 in primary cultures of enteric glia. *P < 0.046, **P = 0.006, ***P < 0.0004, ****P < 0.0001, 2-way ANOVA, Sidak’s multiple comparisons test. N = 6–8 mice. N = 6–7 mice per group.

Article Snippet: One day after the completion of the DSS treatment, mice received an intraperitoneal injection of 100 μg of either a rat anti-mouse CXCL1 (R and D Systems Cat# MAB453, RRID: AB_2087696) or a rat IgG2A isotype control (R and D Systems Cat# MAB006, RRID: AB_357349).

Techniques: Expressing, In Situ, Saline, Incubation, In Vivo, Neutralization, Cell Culture, Inhibition

Journal: Mucosal immunology

Article Title: Enteric glial adenosine 2B receptor signaling mediates persistent epithelial barrier dysfunction following acute DSS colitis.

doi: 10.1038/s41385-022-00550-7

Figure Lengend Snippet: Fig. 7 Summary. During acute intestinal inflammation, glial A2BR signaling decreases the inflammation-induced expression of Csf3, Cxcl1, and Cxcl10, and increases the expression of Il6 by enteric glia. Glial A2BRs in turn cause diverse immunomodulation in the inflamed gut leading to persistent epithelial barrier dysfunction through increased histological damage, overexpression of Cldn1, and localization of the tight junction proteins (TJP).

Article Snippet: One day after the completion of the DSS treatment, mice received an intraperitoneal injection of 100 μg of either a rat anti-mouse CXCL1 (R and D Systems Cat# MAB453, RRID: AB_2087696) or a rat IgG2A isotype control (R and D Systems Cat# MAB006, RRID: AB_357349).

Techniques: Expressing, Over Expression