Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) HBEpC were cultured with cGAMP (14nM), H 2 O 2 , (100μM) or cGAMP+H 2 O 2 for 4 hours. (B-F) qPCR analysis of human IFNα (B) and IFNβ (C) and epithelial cytokines, IL-33 (D), TSLP (E), and RANTES (F). RANTES was stimulated by cGAMP and cGAMP+ H 2 O 2 . IFNβ was also stimulated by cGAMP+H 2 O 2 . Statistical analysis is performed by one-way ANOVA (means ± SEM, n=4). * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) 8 weeks mice were treated with 20 ng of RANTES with 1 μg of HDM or 1 μg of HDM intra nasally on Day 1. Mice were challenged with 1 μg of HDM intranasally on Day 7, then lungs were extracted and analyzed on Day 8. (B) Pictures of lung sections of control and RANTES-adjuvanted, HDM-sensitized mice stained with H-E. Scale bar: 200 μm. Scale bar in picture of high magnification view: 40 μm. (C) Number of cells between bronchus and alveoli analyzed by ImageJ. Statistical analysis is performed by ordinary Mann Whitney’s U test (means ± SEM, 3 points/ section, n=5). (D) Pictures of lung sections in PBS and RANES-adjuvanted, HDM-sensitized mouse stained with PAS/Alcian blue. Scale bar: 80 μm. (E) Ratio of PAS/Alcianble area per epithelial cells (%). **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Control, Staining

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Histopathological analysis of lungs from infected WT and PLG-KO mice inoculated with A/PR/8/34 virus (day 3 post-infection) or A/Netherlands/602/09 virus (day 5 post-infection). Thin sections of lungs obtained from infected and uninfected WT and PLG-KO mice (as indicated) were stained with hematoxilin end eosin (HE) to evaluate histopathological changes. Note the marked infiltration of inflammatory cells in the lungs of infected WT mice, which was largely absent in the lungs of PLG-KO mice. The results shown are representative for two-three mice for both groups. Immunohistochemistry (IHC) using a monoclonal antibody for the influenza A virus nucleoprotein was used to detect virus-infected cells. Cells positive for the presence of viral antigen stained red. (B) Cytokine levels in BAL were assessed by ELISA on the indicated days post inoculation of WT (black bars) and PLG-KO mice (white bars) with IAV A/PR/8/34 or A/Netherlands/602/09. Data represent mean ± s.e.m. of 3–6 mice per group.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Infection, Virus, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Levels of Plasminogen, Active Plasmin, FDP, D-dimer and Fibrinogen, were determined by ELISA in the BAL of A/PR/8/34 infected or uninfected (−) C57BL/6 mice after the indicated days post-inoculation. Markers were also evaluated in the BAL of WT or PLG-KO mice infected with A/Netherlands/602/09 virus. Data represent mean ± s.e.m of n = 3–6 mice per group. (B) Western blot analysis for the detection of fibrinogen and FDP in the lungs of IAV-infected mice on the indicated days post inoculation (representative of n = 3). kDa: apparent molecular weight. n = mice per group. (C) Presence of fibrinogen was assessed in the blood of mice treated or not with Ancrod by ELISA (left panel) or Western blot analysis (right panel). The results represent the mean values ± s.e.m from 3 individual animals per group for the ELISA. The western blot analysis is representative for results of 3 mice per group.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Virus, Western Blot, Molecular Weight

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Survival and weight loss of mice treated with Ancrod (open symbols, n = 11) or not (closed symbols, n = 11) after infection with IAV A/PR/8/34 (squares) or uninfected mice (diamonds, n = 5). Weight loss data represent weight average ± s.e.m of the above indicated number of mice. (B) Cytokines levels in the BAL were measured by ELISA after A/PR/8/34 infection of WT and PLG-KO (KO) mice treated with Ancrod (white bars) or not (black bars). Data represent mean ± s.e.m. of n = 4 mice per group. (C) Survival rate (left panels) and weight loss (right panels) of WT (squares) and PLG-KO (triangles) mice treated with Ancrod (open symbols) or not (closed symbols) after intranasal inoculation with IAV A/PR/8/34 (n = 8–10 mice per group). Weight loss data represent weight average ± s.e.m of the above indicated number of mice.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 3 ADSCs may be affected by endogenous CCL5 signaling. (A) Expression of CCR1 ligands CCL3 and CCL5 in peripheral blood mononuclear cells (BCs), 6 samples of ADSCs (ADSCs 1–6) and skin primary fibroblasts (FBs 1–2) was examined by RT-PCR (CCL3/CCL5 and GAPDH targets were amplified for 40 and 20 cycles, respectively). (B) The level of CCL5 mRNA expression in different samples of ADSCs (I, IV, V, VII, VIII) cultivated for different periods of time (passages p1–p12) was measured using qRT-PCR, normalized with GAPDH expression level and set as 1 in the sample Ip9. The other samples were calculated accordingly. The data are presented as an average mean of one measurement performed in triplicates±SD. Concurrently, concentrations of secreted CCL5 protein (pg/ml) in the conditioning media of the samples I p2, I p9, VIII p2, VII p1, VII p3 and VII p4 were measured by ELISA; NA — not analyzed. (C) CCL5 protein in permeabilized naive or treated with 50 ng/ml of recombinant CCL5 ADSCs (left and right panels, respectively) was examined using CCL5 antibody (red). Nuclear staining was performed using DAPI (blue) (40× magnification). (D) Proliferation and viability of ADSCs cultured in the presence of 25 and 50 ng/ml of CCL5 for 24 and 48 h were tested using ViaLight™plus kit. (E) Three independent lineages of ADSCs were treated with 50 ng/ml of CCL5 for 8, 24, 48 and 72 h, and the level of CCL5 mRNA expression was measured in triplicates, normalized with GAPDH expression level and set as 1 in untreated samples. The data are presented as fold of induction of CCL5 expression in CCL5 treated samples over untreated controls±SD; **pb0.01.

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Staining, Cell Culture

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 4 ADSCs respond to CCL5. (A) ADSCs were stimulated with 50 ng/ml of CCL5 for 3 h (right panel) and subjected to immunostaining using CCR1 antibody (red) and DAPI (blue). Non-stimulated cells are shown in the left panel. (B) ADSCs were treated with different concentrations of CCL5 for 20 min. The levels of phospho-AKT, phospho-ERK, AKT, ERK and GAPDH proteins were detected by WB. (C) CCR1, CCR3 and CCR5 expression levels in two ADSC samples were measured in triplicates by qRT-PCR, normalized with GAPDH mRNA expression levels and set as 1 in sample V (left panel). ADSCs were stimulated with 50 ng/ml of CCL5 for 10 min or 1 h, the cells were lysed and subjected to WB analysis using phospho-ERK, phospho-AKT, ERK, AKT and GAPDH antibodies. (D) Migration capacity of ADSCs towards 50 ng/ml of CCL5 (left panel) was examined using a modified Boyden chamber. As a control, CCL5-free media were used (right panel). The migratory cells were stained and observed under a light microscope using 20× objective.

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Immunostaining, Expressing, Quantitative RT-PCR, Migration, Modification, Control, Staining, Light Microscopy

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 5 CCL5 induces NFκB transcriptional activity. (A) ADSCs were co-transfected with plasmids encoding firefly luciferase reporter under the control of NFκB dependent promoter and Renilla luciferase reporter under SV40 promoter. The cells were stimulated with CCL5 at indicated concentrations for 24 h. Average firefly luciferase activity obtained from three indepen- dent experiments measured in three replicates were normalized to Renilla luciferase values and presented as a fold of induction±SD over luciferase activity in a non-stimulated ADSC sample (set as 1). (B) ADSCs were stimulated with 50 ng/ml of CCL5 for indicated periods of time and several NFκB target gene mRNA expression levels were analyzed by qRT-PCR. Target gene mRNA levels were measured in triplicates and normalized with GAPDH mRNA expression level. The level of the particular gene expression in the untreated control cells at the same time point was set as 1 (indicated by line). Data are represented as an average mean of fold of induction of the indicated gene expression over non- stimulated control±SD obtained from three independent experi- ments. ((A, B) *pb0.05, **pb0.01.)

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Activity Assay, Transfection, Luciferase, Control, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Cell metabolism

Article Title: Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions.

doi: 10.1016/j.cmet.2010.06.008

Figure Lengend Snippet: Figure 1. The Effect of IFNb on Cultured Bone Marrow-Derived Macrophages (A) Treatment of macrophages with IFNb induces IL-10 expression without affecting TNF or IL-12. (B) Uptake of DiI-labeled oxLDL by control (ctrl)- or IFNb-treated macrophages. (C) Uptake of fluorescent latex beads by ctrl- or IFNb-treated macrophages. (D) Surface expression of VLA-4, Mac1, LFA-1, and PSGL1 in ctrl- or IFNb-treated macrophages. (E) Relative gene expression of chemokine receptors in ctrl- or IFNb-treated macrophages. (F) Chemokine expression after IFNb treatment of macrophages. (G) FACS analysis of CCR2 and CCR5 after treatment with IFNb. (H) CCL5 secretion by macrophages after treatment with IFNb. (I) CCL5 expression in ctrl- and IFNb-stimulated IFNAR1WT and IFNAR1del macrophages. (J) CCL5 expression in ctrl- or IFNb-treated wild-type and STAT1/ macrophages. Graphs are representative for at least two independent experiments. Bars represent mean of triplicate wells ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Murine CCL5 secretion from BMM (± 100 U/ml IFNb for 24 hr) was measured by ELISA using anti-mouse CCL5 (R&D Systems, Abigndon, UK) as coating antibody and biotinylated anti-mouse CCL5 (R&D Systems) as detection antibody with mouse CCL5 (Peprotech) as standard.

Techniques: Cell Culture, Derivative Assay, Expressing, Labeling, Control, Gene Expression

Journal: Cell metabolism

Article Title: Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions.

doi: 10.1016/j.cmet.2010.06.008

Figure Lengend Snippet: Figure 3. IFNb Treatment Accelerates Atherosclerosis in apoe/ and ldlr/ Mice (A) Representative lesions of ctrl- or IFNb treated mice of collar-induced atherosclerosis in apoe/ mice. Scale bar, 50 mm. (B) Lesion area measured at six sequential locations proximal from the collar in apoe/ mice that were ctrl or IFNb treated. **p < 0.01 by two-way ANOVA; n = 9/12. (C) Representative lesions in the aortic root of ctrl- or IFNb-treated ldlr/ mice. Scale bar, 200 mm. (D) Lesion area at the aortic root of ctrl- or IFNb-treated ldlr/ mice. *p < 0.05; n = 12/14. (E) Representative MOMA-2-stained lesions from ctrl- and IFNb-treated ldlr/ mice. Scale bar, 100 mm. (F) Absolute macrophage area in lesions from ctrl- or IFNb-treated ldlr/ mice. *p < 0.05; n = 12/14. (G) CD68 expression in aortic arches from ctrl- or IFNb-treated ldlr/ mice. **p < 0.01; n = 11/14. (H) CCL5 levels in plasma from ctrl- or IFNb-treated ldlr/ mice. **p < 0.01; n = 9/11. Shown are mean ± SEM.

Article Snippet: Murine CCL5 secretion from BMM (± 100 U/ml IFNb for 24 hr) was measured by ELISA using anti-mouse CCL5 (R&D Systems, Abigndon, UK) as coating antibody and biotinylated anti-mouse CCL5 (R&D Systems) as detection antibody with mouse CCL5 (Peprotech) as standard.

Techniques: Staining, Expressing, Clinical Proteomics

Journal: Cell metabolism

Article Title: Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions.

doi: 10.1016/j.cmet.2010.06.008

Figure Lengend Snippet: Figure 6. IFNb Induces Chemotactic Factors in Human Primary Macrophages, and Type I IFN Signaling Is Upregulated in Ruptured Human Atherosclerotic Lesions (A) CCR2 expression in macrophages from two independent donors after IFNb treatment. (B) CCR5 expression in macrophages from two independent donors after IFNb treatment. *p < 0.05; **p < 0.01. (C) CCL5 expression in macrophages from two independent donors after IFNb treatment. **p < 0.01. Error bars indicate mean of triplicate wells ± SEM. (D) Ingenuity Pathway Analysis of the differentially expressed genes in stable compared to ruptured carotid endarterectomy specimens. Red signals indicate upregulation and the pathway showed a strongly significant (p = 2.36 3 106; ratio 16/23 [0.696]) upregulation of type I IFN signaling. Indicated are the fold changes (FC) of the respective genes.

Article Snippet: Murine CCL5 secretion from BMM (± 100 U/ml IFNb for 24 hr) was measured by ELISA using anti-mouse CCL5 (R&D Systems, Abigndon, UK) as coating antibody and biotinylated anti-mouse CCL5 (R&D Systems) as detection antibody with mouse CCL5 (Peprotech) as standard.

Techniques: Expressing

Journal: Infection and Immunity

Article Title: Monocyte Chemoattractant Protein 1, Macrophage Inflammatory Protein 1α, and RANTES Recruit Macrophages to the Kidney in a Mouse Model of Hemolytic-Uremic Syndrome

doi: 10.1128/iai.01663-06

Figure Lengend Snippet: FIG. 3. Chemokine mRNA is induced in the kidneys of Stx2- and/or LPS-injected mice. Levels of (A) MCP-1, (B) RANTES, and (C) MIP-1 in mice injected with Stx2 and/or LPS were determined by quantitative real-time RT-PCR. Data are expressed as increase (n- fold) in mRNA over the 72-h time course compared to saline controls (see Materials and Methods for details). Data are representative of two mice per time point, and PCR was performed three times for each chemokine and mouse.

Article Snippet: Other antibodies used were polyclonal rabbit anti-mouse RANTES (Serotec), polyclonal rabbit anti-mouse MIP-1 (Serotec), and polyclonal goat anti-mouse MCP-1 (R&D Systems).

Techniques: Injection, Quantitative RT-PCR, Saline

Journal: Infection and Immunity

Article Title: Monocyte Chemoattractant Protein 1, Macrophage Inflammatory Protein 1α, and RANTES Recruit Macrophages to the Kidney in a Mouse Model of Hemolytic-Uremic Syndrome

doi: 10.1128/iai.01663-06

Figure Lengend Snippet: FIG. 4. Renal chemokine protein is elevated after Stx2 and/or LPS injection. Levels of (A) MCP-1 protein, (B) RANTES protein, and (C) MIP-1 protein in the kidneys of mice injected with Stx2 and/or LPS were determined by ELISA. Chemokine levels were normalized to total kidney protein and are expressed as picograms of chemokine per microgram of total protein. Data are from duplicate experiments and are the averages (SD) from two mice. *, P 0.05 (level in- creased over that of zero hour control); #, P 0.05 (Stx2 plus LPS level increased over that of LPS alone).

Article Snippet: Other antibodies used were polyclonal rabbit anti-mouse RANTES (Serotec), polyclonal rabbit anti-mouse MIP-1 (Serotec), and polyclonal goat anti-mouse MCP-1 (R&D Systems).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Control

Journal: Infection and Immunity

Article Title: Monocyte Chemoattractant Protein 1, Macrophage Inflammatory Protein 1α, and RANTES Recruit Macrophages to the Kidney in a Mouse Model of Hemolytic-Uremic Syndrome

doi: 10.1128/iai.01663-06

Figure Lengend Snippet: FIG. 5. Chemokines are localized to the tubules of kidneys of Stx2 plus LPS-injected mice. Shown are renal MCP-1 immunohistochemistry of mice injected with (A) saline or (B) Stx2 plus LPS (2 h postinjection for the latter), renal RANTES immunohistochemistry of mice injected with (C) saline or (D) Stx2 plus LPS (6 h postinjection for the latter), and renal MIP-1 immunohistochemistry of mice injected with (E) saline or (F) Stx2 plus LPS (2 h postinjection for the latter). The brown color and the arrows indicate positive chemokine staining. Data are representative of three mice. Experiments were performed in duplicate. All images were taken at 400 magnification.

Article Snippet: Other antibodies used were polyclonal rabbit anti-mouse RANTES (Serotec), polyclonal rabbit anti-mouse MIP-1 (Serotec), and polyclonal goat anti-mouse MCP-1 (R&D Systems).

Techniques: Injection, Immunohistochemistry, Saline, Staining

Journal: Infection and Immunity

Article Title: Monocyte Chemoattractant Protein 1, Macrophage Inflammatory Protein 1α, and RANTES Recruit Macrophages to the Kidney in a Mouse Model of Hemolytic-Uremic Syndrome

doi: 10.1128/iai.01663-06

Figure Lengend Snippet: FIG. 6. Chemokine neutralization in mice injected with Stx2 plus LPS inhibits macrophage infiltration and fibrin deposition. Mice were injected with neutralizing antibodies 6 h prior to Stx2 plus LPS injection, and kidneys were removed 48 h after Stx2 plus LPS injection. (A) Inhibition of macrophage recruitment to the kidneys of mice with chemokine-neutralizing antibodies (25 g of each antibody alone or mixed as indicated). Data are percent inhibition of macrophage accumulation compared to the level for the IgG control (25 g rat IgG and 25 g goat IgG)-injected mice. Data are the averages from three to five mice and represent two separate experiments. *, P 0.001 (inhibition greater than that for the first five treatment groups). (B) Fibrin deposition in the glomeruli of mice injected with Stx2 plus LPS and neutralizing antibodies to MCP-1, RANTES, and/or MIP-1 (25 g of each antibody alone or mixed as indicated). Data are the averages from three to five mice and represent two separate experiments. *, P 0.01 (increased over level for the IgG control [25 g rat IgG and 25 g goat IgG]). Also shown is differential staining for fibrin deposition in the glomeruli (arrow) and renal cortex (arrowhead) of mice 48 h after Stx2 plus LPS injection and injection of (C) IgG control antibodies or (D) neutralizing antibodies to MCP-1, RANTES, and MIP-1. Bright red staining indicates fibrin deposition, and red blood cells are stained yellow. Both images were taken at 200 magnification.

Article Snippet: Other antibodies used were polyclonal rabbit anti-mouse RANTES (Serotec), polyclonal rabbit anti-mouse MIP-1 (Serotec), and polyclonal goat anti-mouse MCP-1 (R&D Systems).

Techniques: Neutralization, Injection, Inhibition, Control, Staining

Journal: NPJ Precision Oncology

Article Title: PARP inhibitors promote stromal fibroblast activation by enhancing CCL5 autocrine signaling in ovarian cancer

doi: 10.1038/s41698-021-00189-w

Figure Lengend Snippet: a , b GSEA showing significant enrichment of the inflammatory response and cytokine activity signature in the Ola-treated CAFs. c , d Cytokine profiles from control MRC5-CAFs and MRC5-CAFs primed with either 30 μM Ola or 20 μM Nira for 72 h. The obviously elevated cytokines in the CM of MRC5-CAFs treated with Ola or Nira are listed. e Overlapping cytokines in the CM samples of MRC5-CAFs treated with Ola and Nira. f , g GSEA-derived heatmap showing the relative mRNA expression levels of smooth muscle cell activation-regulated genes in Ola-treated CAFs. h Spearman’s correlation analysis showing the relationship between CCL5 expression and the calculated stromal component score in the TCGA dataset, GSE 9891. i CCL5 mRNA expression by qRT-PCR in primary CAFs with or without PARPi treatment. j ELISA of CCL5 secreted by primary CAFs with or without PARPi exposure. k Immunoblotting of CCL5 protein in primary CAF extracellular conditioned medium (CM) or in cell lysates (IC) after PARPi exposure. GAPDH served as the loading control. Relative densitometric values are labeled in the photographs. l – n Representative IHC images and quantification of CCL5 protein expression in the Ola or Nira-challenged and control xenograft tumor stroma. Measurements were taken from distinct samples ( n = 8). Scale bar, 50 µm. Data are expressed as mean ± s.e.m., * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To verify the effect of CCL5 blockade on stromal activation, we added the combination treatment group of mice that were administered with intraperitoneal Ola (50 mg/kg/day) or Nira (50 mg/kg/day) combining with CCL5 neutralizing antibodies (1 mg/kg, twice a week) (AF-478, R&D Systems, USA).

Techniques: Activity Assay, Control, Derivative Assay, Expressing, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling

Journal: NPJ Precision Oncology

Article Title: PARP inhibitors promote stromal fibroblast activation by enhancing CCL5 autocrine signaling in ovarian cancer

doi: 10.1038/s41698-021-00189-w

Figure Lengend Snippet: a Immunoblotting of α-SMA in primary CAFs after exogenous CCL5 administration. GAPDH served as the loading control. Relative densitometric values are labeled in the photographs. b The CCL5 neutralizing antibody suppressed the α-SMA elevation and cytoskeletal stretch of MRC5-CAFs and primary CAFs in response to Ola. Scale bar, 10 µm. c Representative images and quantification of the collagen contraction assays of MRC-CAFs and primary CAFs under treatment of Ola with or without CCL5 neutralizing antibodies. d Representative images and quantification of CAFs transwell assays using conditional medium derived from CAFs under treatment of Ola with or without CCL5 neutralizing antibodies. e Tumor volumes (mm 3 ) of SKOV3 xenografts estimated using calipers per week for 35 days after tumor cell injections. ( n = 8 mice per group). f Bright field images and weight quantification of subcutaneous tumors from mice ( n = 8) injected intraperitoneally with vehicle or Ola or the combination of Ola and CCL5 neutralizing antibodies for 28 days after the tumor implantation. g Representative images and quantification of Masson’s trichrome staining and picrosirius red staining of the tumor tissue derived from SKOV3 xenografts under the indicated treatment. Measurements were taken from distinct samples ( n = 8). Scale bar, 50 µm. Data are expressed as mean ± s.e.m., * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To verify the effect of CCL5 blockade on stromal activation, we added the combination treatment group of mice that were administered with intraperitoneal Ola (50 mg/kg/day) or Nira (50 mg/kg/day) combining with CCL5 neutralizing antibodies (1 mg/kg, twice a week) (AF-478, R&D Systems, USA).

Techniques: Western Blot, Control, Labeling, Derivative Assay, Injection, Tumor Implantation, Staining

Journal: NPJ Precision Oncology

Article Title: PARP inhibitors promote stromal fibroblast activation by enhancing CCL5 autocrine signaling in ovarian cancer

doi: 10.1038/s41698-021-00189-w

Figure Lengend Snippet: a GSEA of RNA-seq data showing significant enrichment for signature representing the NF-κB pathway activation in Ola-treated CAFs. b Spearman’s correlation analyses showing the relationship between calculated BIOCARTA_NFKB_PATHWAY score and CCL5 expression in the TCGA dataset, GSE 9891, GSE 51088, and GSE 40595. c The luciferase activity of p-NF-κB in CAFs with or without PARPi exposure. d Representative IHC images and quantification of p-P65 protein expression in the Ola-challenged and control tumor stroma of SKOV3 xenografts. Measurements were taken from distinct samples ( n = 8). Scale bar, 50 µm. e The correlation analysis of the IHC scores of CCL5 and p-P65 among SKOV3 tumor xenografts. f Immunoblotting analysis of p-p65 in control and Ola-induced fibroblasts. GAPDH served as the loading control. Relative densitometric values are labeled in the photographs. g Representative immunofluorescence images of p-P65 in CAFs in the presence or absence of Ola. Scale bar, 10 µm. h Immunoblotting analysis of p-P65, CCL5, and α-SMA in control and Ola-challenged fibroblasts in the presence or absence of BAY 117082. GAPDH served as the loading control. Relative densitometric values are labeled in the photographs. i ELISA analysis of CCL5 expression in CM derived from CAFs under the indicated treatment. j , k Representative images and quantification of the collagen contraction assay of CAFs under the indicated treatment. Data are expressed as mean ± s.e.m., * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To verify the effect of CCL5 blockade on stromal activation, we added the combination treatment group of mice that were administered with intraperitoneal Ola (50 mg/kg/day) or Nira (50 mg/kg/day) combining with CCL5 neutralizing antibodies (1 mg/kg, twice a week) (AF-478, R&D Systems, USA).

Techniques: RNA Sequencing, Activation Assay, Expressing, Luciferase, Activity Assay, Control, Western Blot, Labeling, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Derivative Assay, Contraction Assay

Journal: NPJ Precision Oncology

Article Title: PARP inhibitors promote stromal fibroblast activation by enhancing CCL5 autocrine signaling in ovarian cancer

doi: 10.1038/s41698-021-00189-w

Figure Lengend Snippet: a Immunohistochemical detection of α-SMA in matched tumor specimens from patients with OC before and after administration with Ola. Scale bar, 50 µm. b Quantification of α-SMA-positive areas in tumor specimens from OC patients. Same images were measured repeatedly. c – e Representative images and quantification of Masson’s trichrome and picrosirius red staining showing that Ola administration increased the content of stromal components and collagen deposition in human OC tumor specimens. Scale bar, 50 µm. Same images were measured repeatedly. f , g Representative IHC images and quantification of CCL5 and p-P65 in matched tumor specimens from patients with OC before and after administration with Ola. Scale bar, 50 µm. Measures were taken from different samples ( n = 6). h Correlation analysis of CCL5 IHC scores and α-SMA IHC scores in matched tumor specimens from patients with OC before and after administration with Ola. i Correlation analysis of p-P65 IHC scores and CCL5 IHC scores in matched tumor specimens from patients with OC before and after administration with Ola. j Schematic representation of the effect of PARPis on human ovarian stromal fibroblasts. Data are expressed as mean ± s.e.m., * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To verify the effect of CCL5 blockade on stromal activation, we added the combination treatment group of mice that were administered with intraperitoneal Ola (50 mg/kg/day) or Nira (50 mg/kg/day) combining with CCL5 neutralizing antibodies (1 mg/kg, twice a week) (AF-478, R&D Systems, USA).

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Antimitotic chemotherapy promotes tumor NF-kB secretory phenotype and immunosuppressive CXCR2+ neutrophils chemotaxis in triple-negative breast cancers

doi: 10.1101/2024.12.21.629876

Figure Lengend Snippet: A: PCA analysis of 4T1-Luc2 CRISPR CTL and CRISPR STING treated or not by paclitaxel and analyzed by 3’Seq RNA Profiling. B, C: Volcano plots representing differentially up- and downregulated expressed under paclitaxel treatment in 4T1-Luc2 CRISPR CTL and CRISPR STING , respectively. D, E: Signaling pathway signatures up- or downregulated by paclitaxel in 4T1-Luc2 CRISPR CTL and CRISPR STING , respectively. F, G: Concentration in CCL5 and CXCL1 by ELISA in the culture medium of 4T1-Luc2 CRISPR CTL and CRISPR STING treated or not by paclitaxel. H: Concentration in CXCL1 by ELISA spot in the culture medium of paclitaxel-treated 4T1-Luc2 CRISPR CTL and CRISPR STING under treatment with AS602868 or not.

Article Snippet: For ELISA assays, levels of different chemokines in cell supernatants were determined according to the manufacturer’s protocols using following kits: CXCL1 (Mouse CXCL1 Sandwich ELISA Kit, Proteintech) and CCL5 (Mouse CCL5 Sandwich ELISA Kit, Proteintech).

Techniques: CRISPR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Antimitotic chemotherapy promotes tumor NF-kB secretory phenotype and immunosuppressive CXCR2+ neutrophils chemotaxis in triple-negative breast cancers

doi: 10.1101/2024.12.21.629876

Figure Lengend Snippet: A: Impact of STING on paclitaxel-induced transcriptional expression (3’SRP) of Cxcl1, Cxcl5, Ccl5 and Ccl20 comparing fold-change expression (paclitaxel versus untreated) in both CRISPR CTL and CRISPR STING . B: Transcriptional expression fold change of Cxcl1, Cxcl5, Ccl5 and Ccl20 in CRISPR CTL and CRISPR STING under paclitaxel or not. C : Expression of CXCL1 and CCL5 by human TNBC cells using scRNAseq analysis from Broad Institute ( https://singlecell.broadinstitute.org/single_cell/study/SCP1106/stromal-cell-diversity-associated-with-immune-evasion-in-human-triple-negative-breast-cancer ).

Article Snippet: For ELISA assays, levels of different chemokines in cell supernatants were determined according to the manufacturer’s protocols using following kits: CXCL1 (Mouse CXCL1 Sandwich ELISA Kit, Proteintech) and CCL5 (Mouse CCL5 Sandwich ELISA Kit, Proteintech).

Techniques: Expressing, CRISPR