Journal: bioRxiv

Article Title: A Two-Step Activation Mechanism Enables Mast Cells to Differentiate their Response between Extracellular and Invasive Enterobacterial Infection

doi: 10.1101/2023.08.31.555657

Figure Lengend Snippet: A : RNA sequencing of BMMCs left uninfected, or infected with MOI 50 of S. Tm wt or S .Tm Δ invG SL1344 for 4h, presented as the top 30 significantly upregulated genes between S .Tm wt -infected and uninfected control, displayed as a z-score-transformed heatmap. Genes are sorted by the formula “ S .Tm wt - S .Tm Δ invG ” to highlight differences between those two groups. B : Heatmap of relative log2 fold changes between the indicated groups, derived from a cytokine array of 24h supernatants from BMMCs infected with MOI 50 of S. Tm wt or S .Tm Δ invG SL1344. C : Representative IF images of MCs in close contact to CD45+ and CD18+ immune cells in the S. Tm wt SL1344-infected intestinal mucosa and lumen at 48h p.i.. Arrows indicate MCs, scale bars: 10 µm. D : Trypan blue-based live cell counts of bone marrow nucleated cells, cultured for 7 days in base medium supplemented with either medium alone, 24h uninfected BMMC supernatant, S. Tm inoculum-conditioned supernatant, or supernatant of BMMCs infected with S. Tm wt for 24h. E-F : Similar setup as in C, but quantification of the total number of CD45 + CD11b + CD11c − cells (containing monocytes) ( E ) and CD45 + F4/80 + cells (containing macrophages) ( F ) in bone marrow cultures treated with the indicated supernatants, or base medium alone. For C-E, data was statistically analyzed with ANOVA and Dunnet’s posthoc test, using “BMMC+ S .Tm” for comparisons to all other groups.

Article Snippet: Proteome Profiler Mouse XL Cytokine Array (Bio-Techne, #ARY028) was used, following the manufacturer’s instruction for fluorescent detection with IRDye® 800CW Streptavidin (LI-COR, #926-32230) on an Odyssey CLx Infrared Imager.

Techniques: RNA Sequencing Assay, Infection, Transformation Assay, Derivative Assay, Cell Culture

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma

doi: 10.1186/s13046-024-03138-0

Figure Lengend Snippet: GLI1 regulates the expression of CX3CL1. A Representative cytokine array of the supernatants of B16F10 cells transduced with pBABE or pBABE-GLI1. B Expression level of cytokines that exhibited at least 1.5 fold change in pBABE-GLI1 compared to control. Protein expression was assessed by densitometric analysis using ImageJ, and each protein was normalized to array controls. The mean value of the proteins in pBABE was set to 1, and the fold change in pBABE-GLI1 was calculated for each protein. Data represent mean ± SEM. ** p < 0.01; *** p < 0.001 (unpaired Student t test). C, D Western blot of GLI1 in B16F10 and YUMM1.7 cells transduced as indicated. ACTIN was used as loading control. E, F qPCR of Cx3cl1 in B16F10 and YUMM1.7 cells transduced as indicated. Data represent mean ± SEM of three independent experiments. * p < 0.05; *** p < 0.001 (unpaired Student t test). G, H qPCR of GLI1 ( G ) and Cx3cl1 ( H ) in murine allografts injected with B16F10 cell transduced with pBABE or pBABE-GLI1. ** p < 0.01 (unpaired Student t test). I Western blot of GLI1 in YUMM5.2 cells transduced as indicated. ACTIN was used as loading control. J qPCR of Cx3cl1 in YUMM5.2 cells transduced as indicated. Data represent mean ± SEM of three independent experiments. * p < 0.05 (one-way ANOVA). K Western blot of GLI1 in human A375 melanoma cells transduced with pBABE or pBABE-GLI1. L qPCR analysis of CX3CL1 in A375 cells transduced with pBABE or pBABE-GLI1. Data represent mean ± SEM of three independent experiments. ** p < 0.01 (unpaired Student t test). M Schematic representation of the putative GLI consensus site in CX3CL1 promoter. N–O ChIP assay showing that GLI1 and RNA Pol II bind to CX3CL1 promoter in A375 cells. PTCH1 promoter was used as a positive control. The y axis represents the relative promoter enrichment, normalized on the input material. Data represent mean ± SD of three independent experiments. * p < 0.05; **** p < 0.0001 (unpaired Student t test)

Article Snippet: Conditioned media from 48 h incubation of sub-confluent cells in 2.5% FBS-containing media was applied to the Proteome Profiler Human XL Cytokine Array (cat# ARY022B, R&D System) or Proteome Profiler Mouse XL Cytokine Array (cat# ARY028, R&D System) following manufacturer’s instructions.

Techniques: Expressing, Transduction, Control, Western Blot, Injection, Positive Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma

doi: 10.1186/s13046-024-03138-0

Figure Lengend Snippet: CCL7 blockade reverts the activation of moDCs promoted by GLI1 silencing in melanoma cells. A Representative cytokine array in supernatants of SSM2c cells transduced with LV-c or LV-shGLI1. B Expression level of cytokines/chemokines exhibiting at least 1.5 fold change in LV-shGLI1 compared to control (LV-c), which was equated to 1. Protein expression was assessed by densitometric analysis using ImageJ and normalized to array controls. Data represent mean ± SEM. ** p < 0.01; *** p < 0.001 (unpaired Student t test). C Western blot of GLI1 in SSM2c and A375 transduced as indicated. ACTIN was used as loading control. D-E Validation of cytokine array with qPCR of genes shown in ( B ) in SSM2c ( C, D ) and A375 cells ( C, E ) transduced as indicated. Data are expressed as fold change relative to LV-c or pBABE, which were equated to 1. Gene expression is expressed as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. One-way ANOVA ( D ) and unpaired Student t test ( E ). F Representative immunofluorescence images of moDCs cultured 48 h in RPMI media (CTR), CM from SSM2c cells transduced with LV-c or LV-shGLI1 and treated with neutralizing CCL7 antibody or IgG isotype matched control. moDCs were stained with Phalloidin and counterstained with DAPI. Scale bar = 20 μm. G, H Cell circularity (4π*area/perimeter 2 ) ( G ) and cell perimeter (μm) ( H ) analysis of moDCs treated as indicated in ( F ) and quantified by ImageJ. Mean ± SEM from three donors of three independent experiments are reported. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (one-way ANOVA). I Schematic representation of moDC invasion assay. J Invasion assay of moDCs recruited after 48 h by CM from SSM2c cells transduced with LV-c or LV-shGLI1 and treated with blocking CCL7 antibody or IgG isotype. Data represents mean ± SEM of at least three independent experiments. ** p < 0.01; *** p < 0.001; ns, not significant (one-way ANOVA). K Representative pictures of ( J ). DAPI = 4’, 6-diamidino-2-phenylindole

Article Snippet: Conditioned media from 48 h incubation of sub-confluent cells in 2.5% FBS-containing media was applied to the Proteome Profiler Human XL Cytokine Array (cat# ARY022B, R&D System) or Proteome Profiler Mouse XL Cytokine Array (cat# ARY028, R&D System) following manufacturer’s instructions.

Techniques: Activation Assay, Transduction, Expressing, Control, Western Blot, Immunofluorescence, Cell Culture, Staining, Invasion Assay, Blocking Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma

doi: 10.1186/s13046-024-03138-0

Figure Lengend Snippet: An immune signature associated with high GLI and low cytokine expression predicts poor survival in patients with cutaneous melanoma. A Hierarchical clustering of cutaneous melanoma patients based on GLI and cytokine expression ( n = 443 patients, TCGA Pan-Cancer Atlas). B Pearson correlation of RNA expression of GLI1, GLI2 and cytokines CX3CL1, CXCL10, CCL7, CCL2, CXCL1, CCL20, and CXCL8 . C Kaplan–Meier survival curves for probability of survival comparing GLI High/Cytokine Low ( n = 274) (blue line) and GLI Low/Cytokine High ( n = 156) (red line) groups ( p = 0.018)

Article Snippet: Conditioned media from 48 h incubation of sub-confluent cells in 2.5% FBS-containing media was applied to the Proteome Profiler Human XL Cytokine Array (cat# ARY022B, R&D System) or Proteome Profiler Mouse XL Cytokine Array (cat# ARY028, R&D System) following manufacturer’s instructions.

Techniques: Expressing, RNA Expression

Journal: PLoS Biology

Article Title: Mesenchymal Transition and Dissemination of Cancer Cells Is Driven by Myeloid-Derived Suppressor Cells Infiltrating the Primary Tumor

doi: 10.1371/journal.pbio.1001162

Figure Lengend Snippet: (A) Quantification of the subsets of immune cells infiltrating primary tumors and cutaneous metastases. Data were obtained by flow cytometry from 20 cutaneous and 13 eye tumors. Numbers show the frequencies of each subset among CD45 + cells. Gating strategy for immune cell subsets is illustrated in . (B) Preferential accumulation of PMN-MDSC (Gr1 hi F4/80 − ) in primary tumors. Composition of the primary tumor and a cutaneous cheek tumor from the same mouse. Cells are gated on CD45 + CD11c − CD11b + DAPI − tumor cells. (C) Frequency of PMN-MDSC in primary ( n = 13) and cutaneous ( n = 20) tumors. Bars represent mean ± SEM. *** p value <0.0001, two-tailed unpaired Student’s t test. (D) Repertoire of chemokines and cytokines differentially expressed in primary tumors and metastases. Fold changes represent the log 2 ratio of gene expression measured by qRT-PCR in primary tumors ( n = 11) and in the metastatic tumors ( n = 11). p values were calculated by two-tailed paired Student’s t test. (E) Relative expression of CXCL1 (CX1), CXCL2 (CX2), and CXCL5 (CX5) in 11 primary tumors and 11 metastases measured by qRT-PCR. * p <0.05, *** p <0.001. (F–H) CXCL1 (F), CXCL2 (G), and CXCL5 (H) induce PMN-MDSC chemotaxis in vitro, but have no effect on monocytes or T, B, and NK lymphocytes. x -axis represents the amount of chemokine added in ng/ml. Percentage migration was calculated as (number of migrated cells) ×100/(total cells added per well). Data are from three independent experiments carried out in duplicates. Bars represent mean ± SEM * p <0.05, ** p <0.01, *** p <0.001, two-tailed t test. (I) Inhibition of CXCR2 reduces PMN-MDSC migration in vitro. PMN-MDSC were treated 1 h before and during the migration assay with CXCR2 inhibitors—Inh1 (SB265610) and Inh2 (SB225002). Percentage migration was calculated as (number of migrated cells) ×100/(total cells added per well). Data are from two independent experiments carried out in duplicates. Bars represent mean ± SEM * p <0.05, ** p <0.01, two-tailed t test. (J) CXCR2 is required for PMN-MDSC accumulation into the primary tumor. Equal numbers of bone marrow cells from Rosa mT/mG reporter mice expressing tdTomato (used as fluorescently tagged wild-type [ wt ] cells) and Il8rb-KO mice expressing GFP were adoptively transferred into tumor-bearing RETAAD mice. The graph shows the contribution of each genotype to donor-derived PMN-MDSC present in the primary tumor and spleen 18 h after transfer. Data are from four mice. Bars represent mean ± SEM * p <0.05, ** p <0.01, two-tailed t test.

Article Snippet: For each mouse ( n = 11), RNA was extracted from one primary tumor and one cutaneous tumor and gene expression was analyzed using Mouse Common Cytokines PCR Arrays (SABiosciences, PAMM-021) and Mouse Inflammatory Cytokines & Receptors PCR Arrays (SABiosciences, PAMM-011) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Two Tailed Test, Expressing, Quantitative RT-PCR, Chemotaxis Assay, In Vitro, Migration, Inhibition, Derivative Assay

Journal: Communications Biology

Article Title: Macrophage migration inhibitory factor is overproduced through EGR1 in TET2 low resting monocytes

doi: 10.1038/s42003-022-03057-w

Figure Lengend Snippet: a , b Cytokine profile arrays of supernatant collected from cord blood CD34 + cells infected with SCR - and TET2- shRNA-GFP lentiviruses, sorted on GFP expression, and induced to differentiate with stem cell factor (SCF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT3L) and granulocyte-colony stimulating factor (G-CSF). a Representative cytokine array with supernatants collected at day 10 of differentiation. The rectangle points to MIF detection. b Quantification of MIF signals, normalized to positive controls. Data are mean +/− SEM of three independent experiments. Paired t test: * P < 0.05. c MIF concentrations determined by ELISA in the supernatant of cells induced to differentiate for indicated time. Data are mean +/− SEM of indicated independent experiments (day 5: n = 6; day 7: n = 5; day 8: n = 3; day 10: n = 7). Paired t test: * P < 0.05; *** P < 0.001. d RT-qPCR analysis of MIF mRNA expression in four TET2 -depleted ( TET2 shRNA, gray bars) and control ( SCR shRNA, black bars) human leukemic cell lines. Data are mean +/− SEM of three biological replicates. Unpaired t test: * P < 0.05; *** P < 0.001; **** P < 0.0001. e Immunoblot of SCR or TET2 shRNA infected leukemic cell lines sorted on GFP expression. Lower panels, quantification after actin normalization using Image J software. f MIF concentrations determined by ELISA in the supernatants of kasumi-1 ( n = 5), M07e ( n = 5), UT-7 ( n = 4) and TF-1 ( n = 5) cells transduced 24 h before with SCR (black squares) or TET2 (gray squares) shRNA. Data are mean +/− SEM of indicated biological replicates. Unpaired-t test: * P < 0.05; ** P < 0.01. g MIF concentrations determined by ELISA in the plasma of two Tet2 -deficient models (1–3 months): K427 knock-out model (wt/wt or Tet2 +/+ n = 4, wt/exc or Tet2 +/− n = 5 and exc/exc or Tet2 −/− n = 5); ANO knock-down model (wt/wt or Tet2 + /+ n = 5, wt/LacZ or Tet2 +/− n = 5 and LacZ/LacZ or Tet2 −/− n = 6). Data are mean +/− SEM of indicated biological replicates. Dunnett’s multiple comparison tests using wt/wt as control: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD34 + collected supernatants were analyzed using human cytokine antibody array (panel A; R&D Systems).

Techniques: Infection, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Software, Knock-Out, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Systematic Characterization of the Group 2 House Dust Mite Allergen in Dermatophagoides microceras

doi: 10.3389/fcimb.2021.793559

Figure Lengend Snippet: The effects of FIP- fve treatment on the infiltrating inflammatory cells and cytokines in the BALF and the airway inflammation of Der m 2 -sensitized mice. (A) The inflammatory cells in the BALF of Der m 2 -sensitized mice were shown, and the total cells and inflammatory cells were counted (×10 4 ) from the BALF in millimeters by morphometric evaluations of cytospin preparations. (B) The different cytokine sets represent the Th1- and Th2-type responses. Th2-type cytokines were IL-4, IL-5, IL-6, IL-8, and IL-13 in the left panel, and Th1-type cytokines were IL-12, TGF-β, and IFN-γ in the right panel. *** P < 0.001, ** P < 0.01, and * P < 0.05 compared with PC. (C) Histopathological image analysis on the airway inflammation in the lung tissue samples was obtained on day 28 at a magnification of ×100 using a light microscope. NC group, Der m 2 -sensitized/challenged mice (PC), feeding in the first 14 days (pre), and in the last 14 days (post), respectively. All lung tissue was stained with hematoxylin and eosin on the sections to evaluate the inflammation severity and goblet cell hyperplasia (the arrows indicated region). Br, bronchus; Bm, basement membrane; Ep, epithelium.

Article Snippet: The mouse BALF cytokines were analyzed with cytokine array (ARY028, Proteome Profiler Mouse XL Cytokine Array, R&D Systems), according to the manual.

Techniques: Light Microscopy, Staining, Membrane

Journal: Antiviral Research

Article Title: Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin

doi: 10.1016/j.antiviral.2011.02.003

Figure Lengend Snippet: (A) Average levels of the cytokines detected in BALB/c mice infected with a lethal dose of mouse-adapted SARS-CoV on day 3. (B) Average levels of the cytokine RANTES detected in BALB/c mice infected with a lethal dose of mouse-adapted SARS-CoV on day 3. *** p < 0.001, each compound versus PSS.

Article Snippet: Cytokine levels in the supernatant fluids, adjusted for total protein, were measured on the same day using the Quansys Q-PlexTM Mouse Cytokine Array (Quansys Biosciences, Logan, UT).

Techniques: Infection