Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8+ T cells in COPD bronchial inflammatory response

doi: 10.7554/elife.85875

Figure Lengend Snippet: Figure 9. The outcomes of therapeutic interventions are predicted by simulations. (A) Schematic representation of the design used to test therapeutic strategies. Chronic obstructive pulmonary disease (COPD) states were first generated by applying COPD dynamics for 20 years (n=144 simulations). Then, different dynamics were applied for 7 years: COPD dynamics (corresponding to the placebo condition), control dynamics (corresponding to an ideal treatment able to restore all cellular processes, ‘Total inhibition’), and modified COPD dynamics (corresponding to CD54, CXCR1/2, and dual

Article Snippet: DOI: https://doi.org/10.7554/eLife.85875 25 of 57 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- LFA- 1 (mouse monoclonal) BioLegend Cat. #:301233, RRID:AB_2832576 Blocking experiment (1 μg/mL) Antibody Anti- CD54 (mouse monoclonal) Fisher Scientific Cat. #:15247027, RRID:N/A Blocking experiment (10 μg/mL) Antibody Anti- CD86 (mouse monoclonal) Fisher Scientific Cat. #:15297097, RRID:N/A Blocking experiment (10 μg/mL) Antibody Anti- CD44 (rabbit monoclonal) Fisher Scientific Cat. #:15266957, RRID:N/A Blocking experiment (10 μg/mL) Antibody Anti- CXCL8 (mouse monoclonal) BioTechne Cat. #:MAB208- 100, RRID:N/A Blocking experiment (1 μg/mL) Antibody Anti- CD4- PerCP- Vio700 (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 113- 228, RRID:AB_2726039 FC (1:50) Antibody Anti- CD8- PerCP- Vio700 (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 110- 682, RRID:AB_2659249 FC (1:50) Antibody Anti- CD45RA- FITC (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 113- 365, RRID:AB_2726135 FC (1:50) Antibody Anti- granzyme- APC (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 099- 780, RRID:AB_2651900 FC (1:20) Antibody Anti- TNF- α-PE (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 110- 066, RRID:AB_2654213 FC (1:20) Antibody Anti IFN- γ-APC (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 113- 496, RRID:AB_2751119 FC (1:20) Antibody Anti- IL- 17- PE- Cy7 (mouse monoclonal) Miltenyi Biotec Cat. #:130- 120- 413, RRID:AB_2752086 FC (1:20) Antibody Anti- IL- 10- PE (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 112- 728, RRID:AB_2652318 FC (1:20) Antibody Anti- Collagen Type I- FITC (mouse monoclonal) Sigma Aldrich Cat. #:FCMAB412F, RRID:AB_11204160 FC (1:50) Antibody Anti- CD45- APC (mouse monoclonal) BD Pharmingen Cat. #:555485, RRID:AB_398600 FC (1:10) Antibody Anti- CXCR1- PE (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 115- 879, RRID:AB_2727234 FC (1:50) Antibody Anti- CXCR2- APC- Cy7 (human recombinant monoclonal) Miltenyi Biotec Cat. #:130- 119- 571, RRID:AB_2733103 FC (1:50) Continued

Techniques: Generated, Control, Inhibition, Modification

Journal: Cancers

Article Title: Matrix Metalloproteinase 9 Induced in Esophageal Squamous Cell Carcinoma Cells via Close Contact with Tumor-Associated Macrophages Contributes to Cancer Progression and Poor Prognosis.

doi: 10.3390/cancers15112987

Figure Lengend Snippet: Figure 6. IL-8 as a partial inducer of MMP9 in ESCC cells. (A): ELISA quantification of IL-8 in culture supernatants of ESCC cells after direct co-culture with macrophages. Directly co-cultured ESCC cells secreted more IL-8 than monocultured counterparts. (B): Western blot revealed the expression of known IL-8 receptors, CXCR1 (45 kDa, monomer; 80–90 kDa, dimer and their glycosylated forms) and CXCR2 (41 kDa) in TE-9, TE-10, and TE-11 cell lines. (C,D): Treatment with rhIL-8 (100 ng/mL, for 24 h) upregulated MMP9 mRNA expression in all three ESCC cell lines (C) but only triggered MMP9 secretion from TE-10 (D). Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. NS, not significant.

Article Snippet: The primary antibodies used were phosphorylated (p)-Stat3 Tyr705 (#9145, Cell Signaling Technology; CST, Beverly, MA, USA), total (t)-Stat3 (#4904, CST), pAkt Ser473 (#4060, CST), tAkt (#9272, CST), pp38 MAPK (#4511, CST), tp38 MAPK (#8690, CST), CXCR1 (#MAB330, R&D Systems), CXCR2 (#ab65968, Abcam, Cambridge, UK), and β-actin (#4970, CST).

Techniques: Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Cell Culture, Western Blot, Expressing

Journal: Infectious Agents and Cancer

Article Title: HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness

doi: 10.1186/s13027-017-0160-7

Figure Lengend Snippet: Refp17 and vp17s promote breast cancer cells migration. In the wound-healing assay, confluent MDA-MB 231 cell monolayers were serum starved for 24 h and then scratched using a 200 μl pipette tip. Cells were cultured in complete medium either unsupplemented or containing 10 ng/ml of refp17 or vp17s. a After 6 h of culture cells were fixed and stained with Coomassie brilliant blue. The cells migrated into the wound area were counted. b The percentage of wound healing was observed over a period of 12 h. c Wound healing assay was performed pretreating MDA-MB 231 cells for 1 h with a neutralizing mAb to CXCR1 (2.5 μg/ml), CXCR2 (2.5 μg/ml), or with an isotype-matched mAb (Ctrl mAb; 2.5 μg/ml). Images are representative of three independent experiments with similar results (original magnification 10×). Statistical analysis was performed by one-way ANOVA and the Bonferroni’s post-test was used to compare data (** p < 0.01, *** p < 0.001)

Article Snippet: When reported, starved MDA-MB 231 cells were pretreated with 2.5 μg/ml of mAb to CXCR1 (mAb 330; R&D, Minneapolis, MN, USA) or to CXCR2 (mAb 331; R&D), or with an isotype-matched mAb (2.5 μg/ml; R&D) for 1 h at 37 °C before proteins stimulation.

Techniques: Migration, Wound Healing Assay, Transferring, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Role of CX 3 CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

doi: 10.1371/journal.pone.0057230

Figure Lengend Snippet: 500 µl of Matrigel subcutaneously implanted in CX 3 CR1 +/gfp and CX 3 CR1 gfp/gfp mice for 4 weeks. A, Representative sections of Matrigel isolated from CX 3 CR1 +/gfp and CX 3 CR1 gfp/gfp mice, intravitally stained with lectin (Rhodamine Griffonia Simplicifolia ; Red) to show functional MV and DAPI (Nucleus; Blue) show presence of CX 3 CR1 (GFP) positive MV. B, Deficiency of CX 3 CR1 restricted CX 3 CR1 cells to mostly the perivascular region of functional MV within Matrigel. Moreover, the number of integrated vascular associated CX3CR1 positive MV were significantly higher in Matrigels isolated from CX 3 CR1 +/gfp mice compared to Matrigels isolated from CX 3 CR1 gfp/gfp mice. Data is expressed as mean ± SEM of 25 Matrigel sections/mice (n = 3 independently performed experiments). C, Three dimensional reconstruction of the confocal images using IMARIS indicate CX 3 CR1 positive cells integrated into periluminal vascular wall in the Matrigel implanted in CX 3 CR1 +/gfp but not in CX 3 CR1 gfp/gfp mice where cells remained in perivascular region (Scale bar: 10 µm). Microvessels formed in Matrigels isolated from CX 3 CR1 gfp/gfp mice were leaky, which was evident by increased heme content (D & E), Evan's blue dye leak (F & G), and extravasated platelets (CD42b; Red) (H & I) in the matrix compared to Matrigels implanted in CX 3 CR1 +/gfp mice. Data is represented as mean ± SEM of 8 Matrigels/group and 10 Matrigel sections/mice (n = 4 independently performed experiments); * denotes p<0.05.

Article Snippet: For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin (rabbit anti calponin: 1∶200; Epitomics, CA, USA), CX 3 CR1 (rabbit anti CX 3 CR1:1∶50; ProSci Incorporated, CA, USA), F4/80 (Rat anti mouse F4/80: 1∶100; eBioscience; Hatfield, UK), Laminin (mouse anti laminin: 1∶500; Novus Biologicals, Cambridge, UK) and/or CD42b (rat anti CD42b: 1∶200; emfret Analytics GmbH & co.KG, Eibelstadt, Germany).

Techniques: Isolation, Staining, Functional Assay

Journal: PLoS ONE

Article Title: Role of CX 3 CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

doi: 10.1371/journal.pone.0057230

Figure Lengend Snippet: Plaque angiogenesis was created by ligation of carotid artery at its bifurcation for 4 weeks in CX 3 CR1 +/gfp and CX 3 CR1 gfp/gfp mice. Perfusion fixed carotid arteries were isolated and OCT embedded. 5 µm cross sections were stained with smooth muscle marker (Calponin; Red) and confocal images were acquired. A, Representative bright field cross section image of carotid artery from CX 3 CR1 +/gfp mice stained with DAPI (Nucleus; Blue). Red blood cells (RBCs) (arrow heads) were observed in the microvessels indicating these microvessels were functional. B, Mice competent for CX 3 CR1 function had a higher proportion of MV containing RBCs C, Representative cross section image of carotid artery from CX 3 CR1 +/gfp and CX 3 CR1 gfp/gfp mice were stained with calponin (Red) and DAPI (nucleus; blue). CX 3 CR1 positive cells (GFP positive; Green) integrated into microvascular wall and were also present in perivascular region and co-expressed smooth muscle marker (Calponin; Red) (Scale bar: 10 µm). In the CX 3 CR1 functionally deficient (CX 3 CR1 gfp/gfp ) mice the number of vascular and perivascular cells (GFP positive; Green) (D) and co-expressing smooth muscle marker (E) were significantly reduced. Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice (n = 4 independently performed experiments). F, Microvessels in the plaque stained with CX 3 CR1 (Green) and DAPI (nucleus; blue) were 3D reconstructed using IMARIS software to depict signet ring structures and their tubular architecture. G, The number of signet-ring cells was significantly reduced in CX 3 CR1 gfp/gfp mice. H, Schematic representing the three major phenotypes of CX 3 CR1 cells associated with microvascular structures in the plaque. * denotes p<0.05.

Article Snippet: For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin (rabbit anti calponin: 1∶200; Epitomics, CA, USA), CX 3 CR1 (rabbit anti CX 3 CR1:1∶50; ProSci Incorporated, CA, USA), F4/80 (Rat anti mouse F4/80: 1∶100; eBioscience; Hatfield, UK), Laminin (mouse anti laminin: 1∶500; Novus Biologicals, Cambridge, UK) and/or CD42b (rat anti CD42b: 1∶200; emfret Analytics GmbH & co.KG, Eibelstadt, Germany).

Techniques: Ligation, Isolation, Staining, Marker, Functional Assay, Expressing, Software

Journal: PLoS ONE

Article Title: Role of CX 3 CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

doi: 10.1371/journal.pone.0057230

Figure Lengend Snippet: Representative cross sectional images of carotid artery plaques from CX 3 CR1 +/gfp and CX 3 CR1 gfp/gfp mice stained with CD42b (Platelets; Red) (A) or laminin (Basement membrane; Red) (C) and DAPI (Nucleus; Blue). B, Significantly increased staining for platelet CD42b was observed in the neointimal interstitial space in CX 3 CR1 gfp/gfp mice compared to competent CX 3 CR1 +/gfp mice (Scale bar: 50 µm). D, A significantly greater number of CX 3 CR1 positive microvessels were covered by basement membrane laminin in CX 3 CR1 +/gfp mice compared to CX 3 CR1 gfp/gfp mice (insets in panel C show GFP and laminin co-staining present in CX 3 CR1 +/gfp but not CX 3 CR1 gfp/gfp mice). Data is represented as mean ± SEM of 15 plaque sections/mice (n = 4 independently performed experiments); * denotes p<0.05.

Article Snippet: For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin (rabbit anti calponin: 1∶200; Epitomics, CA, USA), CX 3 CR1 (rabbit anti CX 3 CR1:1∶50; ProSci Incorporated, CA, USA), F4/80 (Rat anti mouse F4/80: 1∶100; eBioscience; Hatfield, UK), Laminin (mouse anti laminin: 1∶500; Novus Biologicals, Cambridge, UK) and/or CD42b (rat anti CD42b: 1∶200; emfret Analytics GmbH & co.KG, Eibelstadt, Germany).

Techniques: Staining, Membrane

Journal: PLoS ONE

Article Title: Role of CX 3 CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

doi: 10.1371/journal.pone.0057230

Figure Lengend Snippet: A, Drug (F1) study protocol: Carotid artery was ligated in the C57BL6J mice (wt mice) and animals were treated with a selective CX 3 CR1 inhibitor (F1) (from second week post carotid ligation for another two weeks). B, Representative cross sectional images of carotid artery from C57BL6J mice treated with saline or a selective CX 3 CR1 inhibitor (F1) and stained for laminin (B; Basement membrane; Red), or CD42b (D; Platelets; Red), CX 3 CR1 (GFP; Green) and DAPI (Nucleus; Blue). B & C, Significantly greater number of CX 3 CR1 positive microvessels were covered by basement membrane laminin in saline treated C57BL6J mice compared to F1 treated mice. D & E, Increased platelet CD42b staining was observed in the neointimal interstitial space in the F1 treated mice compared to saline treated mice (Scale bar: 50 µm). In addition the leaky microvessel phenotype in mice treated with F1 was confirmed by presence of intravenously administered (tail vein) 2–2.5 µm diameter microspheres (red spheres) in the neointimal lesion (F & G). IgG control staining for isotype-matched antibodies shown. Data is represented as mean ± SEM of 15 plaque sections/mice (n = 4 independently performed experiments); * denotes p<0.01.

Article Snippet: For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin (rabbit anti calponin: 1∶200; Epitomics, CA, USA), CX 3 CR1 (rabbit anti CX 3 CR1:1∶50; ProSci Incorporated, CA, USA), F4/80 (Rat anti mouse F4/80: 1∶100; eBioscience; Hatfield, UK), Laminin (mouse anti laminin: 1∶500; Novus Biologicals, Cambridge, UK) and/or CD42b (rat anti CD42b: 1∶200; emfret Analytics GmbH & co.KG, Eibelstadt, Germany).

Techniques: Ligation, Saline, Staining, Membrane, Control

Journal: PLoS ONE

Article Title: Role of CX 3 CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

doi: 10.1371/journal.pone.0057230

Figure Lengend Snippet: CX 3 CR1 cells isolated from bone marrow of CX 3 CR1 +/gfp or CX 3 CR1 gfp/gfp mice were sandwiched between two DQ-red fortified (20 µg/ml; substrate for proteases) Matrigel layers with or without 10 ng/ml CX 3 CL1 gradient and were cultured for 5 days. A & B, CX 3 CR1 cells remodelled into tube-like structures (tubulation) especially under CX 3 CL1 gradient (Scale bar: 10 µm). However these tubular structures were incompletely formed by CX 3 CR1 deficient cells. Furthermore, the CX 3 CR1 deficient cells lacked tunneling capacity (dotted lines-protease activation red) in the Matrigel (C & D) and produced significantly less laminin following CX 3 CL1 stimulation (E & F). Data is represented as mean ± SEM of 4 independently performed experiments;* denotes p<0.01.

Article Snippet: For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin (rabbit anti calponin: 1∶200; Epitomics, CA, USA), CX 3 CR1 (rabbit anti CX 3 CR1:1∶50; ProSci Incorporated, CA, USA), F4/80 (Rat anti mouse F4/80: 1∶100; eBioscience; Hatfield, UK), Laminin (mouse anti laminin: 1∶500; Novus Biologicals, Cambridge, UK) and/or CD42b (rat anti CD42b: 1∶200; emfret Analytics GmbH & co.KG, Eibelstadt, Germany).

Techniques: Isolation, Cell Culture, Activation Assay, Produced

Journal: PLoS ONE

Article Title: Role of CX 3 CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

doi: 10.1371/journal.pone.0057230

Figure Lengend Snippet: CX 3 CR1 cells isolated from CX 3 CR1 +/gfp and CX 3 CR1 gfp/gfp mice were treated with 100 nM CX 3 CL1 and 30 minutes post probed for expression of activated RhoA. A, Representative immuno blot showing active RhoA protein expression. B, Active RhoA expression within 30 mins post CX 3 CL1 stimulation was significantly higher in CX 3 CR1 cells isolated from CX 3 CR1 +/gfp compared to CX 3 CR1 gfp/gfp mice. C, CX 3 CR1 cells isolated from bone marrow of CX 3 CR1 +/gfp mice were sandwiched between two Matrigel layers with 10 ng/ml CX 3 CL1 gradient and were cultured for 5 days in absence or presence of Rho inhibitor (Y27632; 10 µM, Scale bar: 10 µm). C & D, Inhibition of Rho using Y27632 (10 µM) prevented the formation of tube-like structures by CX 3 CR1 cells isolated from CX 3 CR1 +/gfp mice. Data is represented as mean ± SEM of 4 independently performed experiments;* denotes p<0.01.

Article Snippet: For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin (rabbit anti calponin: 1∶200; Epitomics, CA, USA), CX 3 CR1 (rabbit anti CX 3 CR1:1∶50; ProSci Incorporated, CA, USA), F4/80 (Rat anti mouse F4/80: 1∶100; eBioscience; Hatfield, UK), Laminin (mouse anti laminin: 1∶500; Novus Biologicals, Cambridge, UK) and/or CD42b (rat anti CD42b: 1∶200; emfret Analytics GmbH & co.KG, Eibelstadt, Germany).

Techniques: Isolation, Expressing, Cell Culture, Inhibition

Journal: Cell death & disease

Article Title: CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation.

doi: 10.1038/s41419-023-06126-y

Figure Lengend Snippet: Fig. 1 CXCR1 deficiency suppresses EAE development. A CXCR1 mRNA expression in peripheral blood leukocytes in healthy controls (n = 21) and MS patients (n = 35) (left panel). Scatterplots showing the correlation between mRNA level of CXCR1 and EDSS in MS patients (right panel). WT and Cxcr1−/−mice were immunized with MOG35–55 peptides in CFA adjuvant and pertussis toxin to induce EAE. B Clinical scores of EAE in immunized WT and Cxcr1−/−mice (left panel), and linear regression analysis (right panel) of the recipient mice depicted (n = 6). The data are expressed as the mean ± SEM. *p < 0.05, vs. WT group (Mann–Whitney U test). C H&E staining and LFB staining of spinal cord paraffin sections from the WT and Cxcr1−/−mice 28 days after EAE induction. Scale bars, 200 μm. D Pooled data are presented from (C). E Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A in CD4+ T cells from the spinal cord and brain of WT and Cxcr1−/−mice on 28 days after EAE induction. Pooled data are presented in the right panel. F ELISA analysis of cytokines (IL-12p70, IL- 6, and TGF-β1) in the serum from WT and Cxcr1−/−mice 12 days after EAE induction. G mRNA levels of Il12a, Il6, and Tgfb1 in the brain from WT and Cxcr1−/−mice on days 0 and 21 after EAE induction. H ELISA analysis of IL-6, IL-12p70, and TGF-β1 in supernatant of DCs sorted from WT and Cxcr1−/−mice 12 days after EAE induction and restimulated with MOG35–55 for 48 h. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT group (one-tailed Student’s t-test). Data are representative of three independent experiments with similar results.

Article Snippet: The following primary antibodies were used: total-AKT (1/1000, CY5561), AKT phosphorylated at Ser473 (1/ 1000, CY6016) and Thr308 (1/1000, CY6017), ERK phosphorylated at T202/Y204 and T185/Y187 (1/1000, CY5277), total-JNK (1/1000, CY5490), GAPDH (1/1000, AB0037) were purchased by Abways Technology; totalERK (1/1000, #9102), total-p38 (1/1000, #8690), p38MAPK phosphorylated at Thr180 and Tyr182 (1/1000, #4511), JNK phosphorylated at Thr183 and Tyr185 (1/1000, #4668), total-S6 (1/1000, #2217), S6 phosphorylated at Ser235/236 (1/1000, #4858) were purchased by Cell Signaling Technology, CXCR1 (1/500, 55450-1-AP) was purchased by Proteintech; anti-HIF-1α (1/1000, SC13515) was purchased by Santa Cruz Biotechnology.

Techniques: Expressing, Adjuvant, MANN-WHITNEY, Staining, Cytometry, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Cell death & disease

Article Title: CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation.

doi: 10.1038/s41419-023-06126-y

Figure Lengend Snippet: Fig. 3 CXCR1 deficiency in DC ameliorates EAE progression. WT or DC conditional knockout mice (DC (WT) or DC (Cxcr1−/−)) were immunized with MOG35–55 peptides in CFA adjuvant and pertussis toxin to induce EAE. A Mean clinical scores of EAE in DC (WT) and DC (Cxcr1−/−) mice (left panel), and linear regression analysis (right panel) of the recipient mice depicted (n = 5). The data are expressed as the mean ± SEM. *p < 0.05, vs. DC (WT) group (Mann–Whitney U test). B Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A in CD4+ T cells from the spinal cord and brain of DC (WT) and DC (Cxcr1−/−) mice 28 days after EAE induction. Pooled data are presented in the right panel. C H&E staining and LFB staining (D) of spinal cord paraffin sections from the DC (WT) and DC (Cxcr1−/−) mice 28 days after EAE induction. Scale bars, 200 μm. Pooled data are presented in the right panel. E IL-12p70, IL-6, and TGF-β1 levels in the serum of DC (WT) and DC (Cxcr1−/−) mice 12 days after EAE induction were detected by ELISA. F Expression of Il12a, Il6, and Tgfb1 mRNA in the spinal cord and brain from the DC (WT) and DC (Cxcr1−/−) mice 28 days after EAE induction. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. DC(WT) group (one-tailed Student’s t-test).

Article Snippet: The following primary antibodies were used: total-AKT (1/1000, CY5561), AKT phosphorylated at Ser473 (1/ 1000, CY6016) and Thr308 (1/1000, CY6017), ERK phosphorylated at T202/Y204 and T185/Y187 (1/1000, CY5277), total-JNK (1/1000, CY5490), GAPDH (1/1000, AB0037) were purchased by Abways Technology; totalERK (1/1000, #9102), total-p38 (1/1000, #8690), p38MAPK phosphorylated at Thr180 and Tyr182 (1/1000, #4511), JNK phosphorylated at Thr183 and Tyr185 (1/1000, #4668), total-S6 (1/1000, #2217), S6 phosphorylated at Ser235/236 (1/1000, #4858) were purchased by Cell Signaling Technology, CXCR1 (1/500, 55450-1-AP) was purchased by Proteintech; anti-HIF-1α (1/1000, SC13515) was purchased by Santa Cruz Biotechnology.

Techniques: Knock-Out, Adjuvant, MANN-WHITNEY, Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Expressing, One-tailed Test

Journal: Cell death & disease

Article Title: CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation.

doi: 10.1038/s41419-023-06126-y

Figure Lengend Snippet: Fig. 4 Inhibition of CXCL5 action attenuates EAE progression. A Schematic diagram of EAE mice treated with CXCL5 neutralizing antibody (anti- CXCL5) or isotype control (IgG). B Clinical scores (left panel) and linear regression analysis (right panel) of the recipient mice. The data are expressed as the mean ± SEM (n = 7). **p < 0.01, ***p < 0.001 vs. IgG group (Mann–Whitney U test). C LFB staining and D H&E staining of spinal cord paraffin sections from the IgG and anti-CXCL5 (5 μg) treated mice 28 days after EAE induction. Pooled data are presented in the right panel. E Pooled data are presented from (C, D). F Frequencies of CD4+ T cells and G expression of IFN-γ and IL-17A from the spinal cord and brain 28 days after EAE induction. H Pooled data are presented from (G). *p < 0.05, **p < 0.01, ***p < 0.001 vs. IgG group (one-tailed Student’s t-test). I Schematic diagram of DC (WT) or DC (Cxcr1−/−) mice that were induced EAE and treated with 1.6 μg anti-CXCL5 or IgG. J Clinical scores (left panel) and linear regression analysis (right panel) of the recipient mice. The data are expressed as the mean ± SEM (n = 7). **p < 0.01, ***p < 0.001 vs. IgG group (Mann–Whitney U test).

Article Snippet: The following primary antibodies were used: total-AKT (1/1000, CY5561), AKT phosphorylated at Ser473 (1/ 1000, CY6016) and Thr308 (1/1000, CY6017), ERK phosphorylated at T202/Y204 and T185/Y187 (1/1000, CY5277), total-JNK (1/1000, CY5490), GAPDH (1/1000, AB0037) were purchased by Abways Technology; totalERK (1/1000, #9102), total-p38 (1/1000, #8690), p38MAPK phosphorylated at Thr180 and Tyr182 (1/1000, #4511), JNK phosphorylated at Thr183 and Tyr185 (1/1000, #4668), total-S6 (1/1000, #2217), S6 phosphorylated at Ser235/236 (1/1000, #4858) were purchased by Cell Signaling Technology, CXCR1 (1/500, 55450-1-AP) was purchased by Proteintech; anti-HIF-1α (1/1000, SC13515) was purchased by Santa Cruz Biotechnology.

Techniques: Inhibition, Control, MANN-WHITNEY, Staining, Expressing, One-tailed Test

Journal: Cell death & disease

Article Title: CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation.

doi: 10.1038/s41419-023-06126-y

Figure Lengend Snippet: Fig. 6 Deletion of CXCR1 protects from LPS-induced acute lung injury. A Percent survival of WT and Cxcr1−/−mice after LPS (10 mg/kg) intraperitoneal compared with saline controls. (n = 8, Kaplan–Meier survival curves/Mantel–Cox analysis). B H&E staining images of lung tissue from WT and Cxcr1−/−mice after saline or 10 mg/kg LPS challenge. Tissue was collected 24 h after administration. C–E Bronchoalveolar lavage fluid (BALF) was obtained after WT and Cxcr1−/−mice 24 h intraperitoneal injection of LPS (10 mg/kg). Representative flow cytometry data showing frequencies of CD4+ T cells (C) IFN-γ+ and IL-17A+ (D). Pooled data are presented in the right panel. E ELISA analysis of IL-6, IL-12p70, and CXCL5 concentrations in BALF. F, G Immune cells were sorted from the lung tissue, and the percentage of CD4+ T cells (F), IFN-γ, and IL- 17A in CD4+ T (G) were analyzed by flow cytometry. Pooled data are presented in the right panel. H Quantitation of Il6, Il12a, Cxcl5, and Hif1α mRNA level in lung tissue of WT and Cxcr1−/−mice stimulated with LPS for 24 h. Data are mean ± SEM (n = 5–6 in B–E, G, H). *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT group (one-tailed Student’s t-test). Data are representative of three independent experiments with similar results.

Article Snippet: The following primary antibodies were used: total-AKT (1/1000, CY5561), AKT phosphorylated at Ser473 (1/ 1000, CY6016) and Thr308 (1/1000, CY6017), ERK phosphorylated at T202/Y204 and T185/Y187 (1/1000, CY5277), total-JNK (1/1000, CY5490), GAPDH (1/1000, AB0037) were purchased by Abways Technology; totalERK (1/1000, #9102), total-p38 (1/1000, #8690), p38MAPK phosphorylated at Thr180 and Tyr182 (1/1000, #4511), JNK phosphorylated at Thr183 and Tyr185 (1/1000, #4668), total-S6 (1/1000, #2217), S6 phosphorylated at Ser235/236 (1/1000, #4858) were purchased by Cell Signaling Technology, CXCR1 (1/500, 55450-1-AP) was purchased by Proteintech; anti-HIF-1α (1/1000, SC13515) was purchased by Santa Cruz Biotechnology.

Techniques: Saline, Staining, Injection, Cytometry, Enzyme-linked Immunosorbent Assay, Quantitation Assay, One-tailed Test

Journal: Cell death & disease

Article Title: CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation.

doi: 10.1038/s41419-023-06126-y

Figure Lengend Snippet: Fig. 7 CXCR1 governs the pathogenesis of LPS-induced acute lung injury in DCs. A Percent survival of DC(WT) and DC(Cxcr1−/−) mice after LPS (10 mg/kg) intraperitoneal compared with saline controls. (n = 8, Kaplan–Meier survival curves/Mantel–Cox analysis). B H&E staining images of lung tissue from DC(WT) and DC(Cxcr1−/−) mice after saline or 10 mg/kg LPS challenge. Tissue was collected 24 h after administration. C–E Bronchoalveolar lavage fluid (BALF) was obtained after DC(WT) and DC(Cxcr1−/−) mice 24 h intraperitoneal injection of LPS (10 mg/kg). Representative flow cytometry data showing frequencies of CD4+ T cells (C) IFN-γ+ and IL-17A+ (D). Pooled data are presented in the right panel. E ELISA analysis of IL-6, IL-12p70, and CXCL5 concentrations in BALF. F, G Immune cells were sorted from the lung tissue, and the percentage of CD4+ T cells (F) and the proportion of IFN-γ and IL-17A in CD4+ T cells (G) were analyzed by flow cytometry. Pooled data are presented in the right panel. H Quantitation of Il6, Il12a, and Hif1α mRNA levels in lung tissue of DC(WT) and DC(Cxcr1−/−) mice stimulated with LPS for 24 h. Data are mean ± SEM (n = 5–6 in B–E, G, H). *p < 0.05, **p < 0.01, ***p < 0.001 vs. DC (WT) group (one-tailed Student’s t-test). Data are representative of three independent experiments with similar results.

Article Snippet: The following primary antibodies were used: total-AKT (1/1000, CY5561), AKT phosphorylated at Ser473 (1/ 1000, CY6016) and Thr308 (1/1000, CY6017), ERK phosphorylated at T202/Y204 and T185/Y187 (1/1000, CY5277), total-JNK (1/1000, CY5490), GAPDH (1/1000, AB0037) were purchased by Abways Technology; totalERK (1/1000, #9102), total-p38 (1/1000, #8690), p38MAPK phosphorylated at Thr180 and Tyr182 (1/1000, #4511), JNK phosphorylated at Thr183 and Tyr185 (1/1000, #4668), total-S6 (1/1000, #2217), S6 phosphorylated at Ser235/236 (1/1000, #4858) were purchased by Cell Signaling Technology, CXCR1 (1/500, 55450-1-AP) was purchased by Proteintech; anti-HIF-1α (1/1000, SC13515) was purchased by Santa Cruz Biotechnology.

Techniques: Saline, Staining, Injection, Cytometry, Enzyme-linked Immunosorbent Assay, Quantitation Assay, One-tailed Test

Journal: Cell death & disease

Article Title: CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation.

doi: 10.1038/s41419-023-06126-y

Figure Lengend Snippet: Fig. 8 Graphical summary of CXCR1 drives dendritic cell-dependent inflammation and autoimmune pathogenesis. LPS or virus infection upregulates the level of HIF-1α via CXCR1 and Erk signaling pathways in DCs. The increasing level of HIF-1α binds to the CXCL5 promoter and causes an upregulation of CXCL5 expression, then forming a CXCL5/CXCR1/HIF-1α positive feedback loop. This feedback loop promotes IL-6 and IL-12p70 expression, ultimately leading to inflammation damage in the EAE and ARDS mice model. LPS lipopolysaccharide, DC dendritic cells, ARDS acute respiratory distress syndrome.

Article Snippet: The following primary antibodies were used: total-AKT (1/1000, CY5561), AKT phosphorylated at Ser473 (1/ 1000, CY6016) and Thr308 (1/1000, CY6017), ERK phosphorylated at T202/Y204 and T185/Y187 (1/1000, CY5277), total-JNK (1/1000, CY5490), GAPDH (1/1000, AB0037) were purchased by Abways Technology; totalERK (1/1000, #9102), total-p38 (1/1000, #8690), p38MAPK phosphorylated at Thr180 and Tyr182 (1/1000, #4511), JNK phosphorylated at Thr183 and Tyr185 (1/1000, #4668), total-S6 (1/1000, #2217), S6 phosphorylated at Ser235/236 (1/1000, #4858) were purchased by Cell Signaling Technology, CXCR1 (1/500, 55450-1-AP) was purchased by Proteintech; anti-HIF-1α (1/1000, SC13515) was purchased by Santa Cruz Biotechnology.

Techniques: Virus, Infection, Protein-Protein interactions, Expressing