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: Journal of translational medicine

Article Title: RBM15 recruits myeloid-derived suppressor cells via the m6A-IGF2BP3/CBR3-AS1/miR-409-3p/CXCL1 axis, facilitating radioresistance in non-small-cell lung cancer.

doi: 10.1186/s12967-025-06205-y

Figure Lengend Snippet: Fig. 4 CBR3-AS1 recruits MDSCs through the miR-409-3p/CXCL1/CXCR2 axis. A Relative mRNA expression levels of CXCL1 in H520 cells transfected with a control empty vector (vecCtrl), miR-409-3p mimic plasmid (miR-409-3p), control shRNA (shCtrl), or specific interfering hairpin RNAs targeting miR-409-3p (shmiR-409-3p). The cells were exposed to radiation after 24 h of transfection. B Relative protein expression levels of CXCL1 in H520 cells. β-actin was used as the reference for normalization. C Relative luciferase activity of CXCL1 under miR-409-3p overexpression in the wild-type and mutant CXCL1 groups treated with radiation. D, E Relative mRNA and protein expression levels of CXCL1 in H520 cells transfected with shCtrl, shCBR3-AS1, or shmiR-409-3p. F Migration ability of MDSCs toward the conditioned medium of H520 cells transfected with CXCL1, shCXCL1, or treated with a CXCR2 inhibitor (SB265610, 10 mM) and treated with radiation. G, H Levels of interferon (IFN)-γ secreted by CD8+ and CD4.+ T cells co-cultured with H520 cells transfected with CXCL1, shCXCL1 or treated with a CXCR2 inhibitor (SB265610, 10 mM). IFN-γ expression was reported as the mean fluorescence intensity. The significance between two groups was analyzed using Student’s t-test. Data are presented as mean ± SD from three independent experiments. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Membranes were probed with primary antibodies: RBM15 (1:500, Abclonal, USA, A4936), IGF2BP3 (1:1000, Abclonal, A4444), CXCL1 (1:500, Abclonal, USA, A5802), CXCR2 (1:1000, Proteintech, Wuhan, China, 20634-1-AP), or β-actin (1:500, Abclonal, USA, WL01372).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, shRNA, Luciferase, Activity Assay, Over Expression, Mutagenesis, Migration, Cell Culture, Fluorescence

Journal: Veterinary Research

Article Title: Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome

doi: 10.1186/s13567-014-0113-8

Figure Lengend Snippet: Standards, capture and detection antibodies used by FMIA and ELISA

Article Snippet: IL8 (27) , SD061 (L) , MCA1660 (C) , MAB5351 (RD).

Techniques: Enzyme-linked Immunosorbent 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: 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: BMC Genomics

Article Title: Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome

doi: 10.1186/s12864-016-2565-x

Figure Lengend Snippet: IL8 protein production from Holstein and Angus fibroblasts. a IL8 production by dermal fibroblasts isolated from 19-month old Holstein ( n = 5) and Angus ( n = 12) heifers and challenged for 24 h with LPS (100ng/ml) or IL1B (1ng/ml). b Eight cultures ( n = 4/breed) were randomly chosen for RNA-seq and MIRA-seq analysis and levels of IL8 protein following a second 24 h challenge with either LPS (100ng/ml) or IL1B (1ng/ml) on these 8 cultures are shown. There was no detectable IL8 production from un-treated cultures of both breeds. Values are mean ± SEM. * indicates P < 0.05, ** indicates P < 0.01

Article Snippet: Mouse anti-bovine (clone 170.13, gifted by Samuel Maheswaren, University of Minnesota, St. Paul, MN) and a biotinylated goat anti-human IL8 (R&D Systems Inc., Minneapolis, MN) were used as capture and detection antibodies, respectively.

Techniques: Isolation, RNA Sequencing

Journal: BMC Genomics

Article Title: Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome

doi: 10.1186/s12864-016-2565-x

Figure Lengend Snippet: Average response to LPS from combined Holstein and Angus cultures measured by increases in expression of immune-associated genes compared to hour 0 post-LPS

Article Snippet: Mouse anti-bovine (clone 170.13, gifted by Samuel Maheswaren, University of Minnesota, St. Paul, MN) and a biotinylated goat anti-human IL8 (R&D Systems Inc., Minneapolis, MN) were used as capture and detection antibodies, respectively.

Techniques: Expressing, Activation Assay, Ubiquitin Proteomics, Virus

Journal: BMC Genomics

Article Title: Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome

doi: 10.1186/s12864-016-2565-x

Figure Lengend Snippet: Differential gene expression (fold change) of immune-associated genes at hours 0, 2, and 8 post-LPS treatment of fibroblasts collected from Holstein animals as compared to fibroblasts collected from Angus animals

Article Snippet: Mouse anti-bovine (clone 170.13, gifted by Samuel Maheswaren, University of Minnesota, St. Paul, MN) and a biotinylated goat anti-human IL8 (R&D Systems Inc., Minneapolis, MN) were used as capture and detection antibodies, respectively.

Techniques: Gene Expression, DNA Methylation Assay

Journal: BMC Genomics

Article Title: Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome

doi: 10.1186/s12864-016-2565-x

Figure Lengend Snippet: Selected gene expression by Holstein and Angus fibroblasts in response to LPS. Differences in expression of TLR4 ( a ) IL8 ( b ) TNF ( c ) CCL20 ( d ) and CCL5 ( e ) between Holstein and Angus fibroblast cultures at hours 0, 2, and 8 post-LPS exposure were determined by RT-qPCR. Values are expressed as delta Ct, or the difference in cycles to threshold (Ct) between the gene of interest and the endogenous gene control beta-actin ( ACTB) . Fold differences (2 -ΔΔCt ) in expression between Holstein and Angus cultures are indicated in parentheses. All values are mean ± SEM. ( n = 4/group). * P < 0.05 and ** P < 0.01 ( t -test)

Article Snippet: Mouse anti-bovine (clone 170.13, gifted by Samuel Maheswaren, University of Minnesota, St. Paul, MN) and a biotinylated goat anti-human IL8 (R&D Systems Inc., Minneapolis, MN) were used as capture and detection antibodies, respectively.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Control

Journal: BMC Genomics

Article Title: Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome

doi: 10.1186/s12864-016-2565-x

Figure Lengend Snippet: RT-qPCR primer pairs used for amplification of target genes

Article Snippet: Mouse anti-bovine (clone 170.13, gifted by Samuel Maheswaren, University of Minnesota, St. Paul, MN) and a biotinylated goat anti-human IL8 (R&D Systems Inc., Minneapolis, MN) were used as capture and detection antibodies, respectively.

Techniques: Amplification, Sequencing