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

Article Title: Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases

doi: 10.1073/pnas.1220580110

Figure Lengend Snippet: Tumor perfusion and changes in expression of CXCL12, VEGF, as well as numbers of CEPs and neutrophils/G-MDSCs induced in tumor after oncolytic virotherapy treatment. (A) Tumor-bearing mice were injected intravenously with 100 μL of a 50% solution of 100-nm-diameter orange fluorescent microspheres. Five minutes later, animals were killed and tumors were immediately snap-frozen for analyses of tumor perfusion by visualizing fluorescent microspheres in the vasculature of fixed sections using a Zeiss Axiophot HRM Inverted fluorescent microscope and analyzed using Image-Pro-6.2 software. (Scale bars, 100 μm.) (B) Intratumoral expression of CXCL12 was determined in tumor stromal cell-enriched supernatants derived from tumors resected on day 8 after the treatment, whereas expression of VEGF (C) was determined in tumor lysates as described in Materials and Methods. ELISAs were performed on media or lysates and colorimetric values were measured by microplate reader at 450 nm. (D) Recruitment of CEPs (CD45-c-kit+VEGFR-2+) was determined in control and virally treated tumors with 8 d after treatment. Single-cell suspensions were prepared from 4T1 tumors and stained with anti–CD45-APC-Cy7, anti–VEGFR-2-PerCP-Cy5.5, and anti–c-kit-PE mAbs. (E) The percentage of mobilization neutrophils/G-MDSCs in control and treatment groups. Cells were stained with anti-CD11b-APC, anti-Ly6C-FITC, and anti–Ly6G-PE mAbs and analyzed by flow cytometry. Background staining was assessed using isotype control antibodies. Results are presented as the means ± SD of three or four independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The cells were plated on tissue culture plates and cultured for 48 h. Media containing equal amount of protein (100 μg) were analyzed in ELISA by using CXCL12/SDF-1α Elisa Quantikine kit (R&D Systems).

Techniques: Expressing, Injection, Microscopy, Software, Derivative Assay, Staining, Flow Cytometry

Journal: Cell host & microbe

Article Title: Staphylococcus aureus virulent PSMα peptides induce keratinocyte alarmin release to orchestrate IL-17-dependent skin inflammation

doi: 10.1016/j.chom.2017.10.008

Figure Lengend Snippet: A, WT mice treated with IL-1α blocking antibody (IL-1αAb) or isotype-matched control Ab were epicutaneously colonized with S. aureus. Representative macroscopic images of mice colonized with S. aureus (n=5 mice per group). B–C, Skin disease scores (B) and S. aureus CFU in the skin (C) of WT mice treated with IL-1α Ab or control Ab. Each dot represents a mouse. D, Skin tissues of WT, and Myd88Δker and Myd88−/− mice colonized with S. aureus or treated with PBS were stained with Hoechst stain (blue) and antibody against IL-1α (red) or Hoechst stain (blue) and antibodies against S. aureus (red) and IL-36α (green). Scale bars, 50 μm. E, The numbers of S. aureus per high power field are (HPF) are shown. Each dot represents average results from an individual mouse. Data are representative of 2 independent experiments. F–K, IL-1α (F, H, J) and IL-36α (G, I, K) release of differentiated primary KCs isolated from WT and Myd88−/− mice (F, G), WT and Ilr1−/− mice (H, I), or WT mice in the presence of anti-IL-36 neutralizing Mab or isotype-matched control Mab (J, K) and stimulated with culture supernatants of S. aureus for indicated time. IL-1α and IL-36α were detected by ELISA assay and immunoblotting, respectively. β-actin in whole cell lysates is shown as loading control. Data are presented as mean±SD. Data are presented as mean ± SD. Data are representative of at least 2 independent experiments. ND; not detected, n.s.; not significant, *P<0.05 and **P<0.01, by unpaired two-tailed Mann-Whitney U test (B, C, F, H, J) or Kruskal-Wallis test (E).

Article Snippet: Human IL-1α was detected with Quantikine ® ELISA Human IL-1α/IL-1F1 Immunoassay (R&D).

Techniques: Blocking Assay, Staining, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell host & microbe

Article Title: Staphylococcus aureus virulent PSMα peptides induce keratinocyte alarmin release to orchestrate IL-17-dependent skin inflammation

doi: 10.1016/j.chom.2017.10.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human IL-1α was detected with Quantikine ® ELISA Human IL-1α/IL-1F1 Immunoassay (R&D).

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Software, FCAP Assay

Journal: International Journal of Oncology

Article Title: Cancer-associated fibroblast-induced M2-polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor-1 pathway

doi: 10.3892/ijo.2021.5239

Figure Lengend Snippet: PAI-1 is the key factor secreted by TAMs following CAF-CM stimulation and promotes tumor malignant behavior in vitro . (A) Original image and (B) spot pixel value from a cytokine array revealed the profiles of paracrine factors in the M0-CM, TAM (Ca)-CM and TAM(CAF)-CM. (C) Western blot and (D) ELISA results indicated that PAI-1 was upregulated in the TAM(CAF) group compared with that in the M0 and TAM(Ca) groups. (E) Cell Counting Kit-8 and (F) colony formation assays showed that the inhibition of PAI-1 in TAM(CAF)-CM suppressed the enhanced proliferation of Huh-7 cells. (G) Wound healing (scale bar, 400 µ m) and (H) Transwell and (I) Matrigel (scale bar, 200 µ m) assays indicated that the inhibition of PAI-1 in the TAM(CAF)-CM group suppressed the enhanced migration and invasion of the Huh-7 cells. * P<0.05; ** P<0.01. CAF, cancer-associated fibroblast; CM, conditioned medium; CXCL12, C-X-C motif chemokine ligand 12; CXCL5, C-X-C motif chemokine ligand 5; PAI-1, plasminogen activator inhibitor-1; PDGF-AA, platelet derived growth factor-AA; TAM, tumor-associated macrophage; TIM-3, T-cell immunoglobulin mucin 3; M0, macrophages; NS, not significant.

Article Snippet: The Human IL-6 Quantikine ELISA kit (cat. no. D6050), Human Serpin E1/PAI-1 Quantikine ELISA kit (cat. no. DSE100) and Human CXCL12/stromal cell-derived factor 1α Quantikine ELISA kit (cat. no. DSA00) were purchased from R&D Systems Inc., to detect the concentrations of IL-6, PAI-1 and CXCL12 in the CM, according to the manufacturer's instructions.

Techniques: In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Inhibition, Migration, Derivative Assay

Journal: International Journal of Oncology

Article Title: Cancer-associated fibroblast-induced M2-polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor-1 pathway

doi: 10.3892/ijo.2021.5239

Figure Lengend Snippet: CAF-derived CXCL12 induces the secretion of PAI-1 in TAM(CAF). (A) Original image and (B) spot pixel value from a cytokine array to identify the different patterns of molecules in cancer-CM and CAFs-CM. CXCL12 gene expression and its secretion were increased in CAFs compared with that in the Huh-7 and Lx-2 cells following (C) RT-qPCR and (D) ELISA. (E) CXCR4 gene expression in M0, TAM(Ca) and TAM(CAF) was analyzed using RT-qPCR. (F) RT-qPCR and (G) ELISA results demonstrated that the gene expression level and secretion of PAI-1 were decreased in TAM(CAF) after CXCL12 neutralization in CAF-CM, respectively. (H) Schematic representation of the proposed interactions between HCC, TAMs and CAFs. * P<0.05; ** P<0.01. CAF, cancer-associated fibroblast; CM, conditioned medium; CCL5, C-C motif chemokine ligand 5; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C Motif chemokine receptor 4; HCC, hepatocellular carcinoma; HSCs, hepatic stellate cells; PAI-1, plasminogen activator inhibitor-1; PDGF-AA, platelet derived growth factor-AA; RT-qPCR, reverse transcription-quantitative PCR; TAM, tumor-associated macrophage; Ab, antibody.

Article Snippet: The Human IL-6 Quantikine ELISA kit (cat. no. D6050), Human Serpin E1/PAI-1 Quantikine ELISA kit (cat. no. DSE100) and Human CXCL12/stromal cell-derived factor 1α Quantikine ELISA kit (cat. no. DSA00) were purchased from R&D Systems Inc., to detect the concentrations of IL-6, PAI-1 and CXCL12 in the CM, according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Neutralization, Reverse Transcription, Real-time Polymerase Chain Reaction