Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: Lipocalin-2 mediates C/EBPβ-driven CTLA-4 up-regulation in T cells. (A) Venn diagram of genes up-regulated in CEBPB -oe compared to EV cells in CT26, SNU1544, and SNU81, analyzed by RNA sequencing, identifying 13 genes that were concurrently up-regulated. (B) CM from EV or Cebpb -oe CT26 cells were subjected to mouse cytokine array. Each cytokine is represented by duplicate spots, and those showing altered expression are indicated (left). The graph shows signal intensity relative to control spots, analyzed by densitometric quantification (right). (C) Protein levels of C/EBPβ and lipocalin-2 in SNU1544 and SW837 cells by Western blotting . (D) mRNA expression of CTLA4 in Jurkat cells after 24 h co-culture with indicated cells, measured by real-time PCR. (E) mRNA expression of immune checkpoint genes in Jurkat cells after co-culture with LCN2 -oe or EV SNU1544 cells. (F) mRNA expression of CTLA4 in Jurkat cells co-cultured with SW837 cells at a 1:1 ratio in the presence of lipocalin-2 neutralizing antibody (1 μg/ml) or isotype IgG control for 24 h. (G) Protein levels of CEBP/β and lipocalin-2 in CEBPB -oe and EV SNU1544 cells (left). mRNA levels of CTLA4 in Jurkat cells co-cultured with CEBPB -oe or EV SNU1544 cells in the presence of lipocalin-2 neutralizing antibody (1 μg/ml) or isotype IgG control for 24 h (right). (H) Protein levels of C/EBPβ and lipocalin-2 in EV, Cebpb -oe, and Lcn2 -oe CT26 cells (left). Flow cytometry analysis of CTLA-4 + in CD4 + and CD8 + cells from activated mouse splenic T cells co-cultured with EV or Lcn 2-oe CT26 cells for 72 h at the indicated culture ratio of T cells to CT26 cells (right). (I) Flow cytometry analysis of activated mouse splenic T cells co-cultured with Cebpb -oe CT26 at a 5:1 ratio for 48 h in the presence of lipocalin-2 neutralizing antibody (2 μg/ml) or isotype IgG control. The bar graphs represent the mean ± SD; each dot indicates a biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: AKR1C1 , aldo-keto reductase family 1 member C1; C3 , complement C3; CDA , cytidine deaminase; CD14 , cluster of differentiation 14; CEBPB , CCAAT enhancer binding protein beta; CEBPB -oe, CEBPB overexpression; CM, conditioned media; CTLA4 , cytotoxic T-lymphocyte associated protein 4; CXCL1, C-X-C motif chemokine ligand 1; EV, empty vector; IL1RL1 , interleukin 1 receptor like 1; IRF7 , interferon regulatory factor 7; LCN2 , lipocalin-2; Lcn2 -oe, Lcn2 -overexpression; S100A8 , S100 calcium binding protein A8; S100A9 , S100 calcium binding protein A9; SBSN , suprabasin; SD, standard deviation; SERPINB2 , serpin family B member 2; SLPI , secretory leukocyte peptidase inhibitor.

Article Snippet: CM was concentrated using an Amicon Ultra centrifugal filter 3K device (UFC500324, MilliporeSigma) and applied to a mouse cytokine array (ARY028, R&D Systems), according to the manufacturer’s protocol, to assess the levels of 111 cytokines and chemokines.

Techniques: RNA Sequencing, Expressing, Control, Western Blot, Co-Culture Assay, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Binding Assay, Over Expression, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Neurology

Article Title: Inhibition of Colony Stimulating Factor 1 Receptor Suppresses Neuroinflammation and Neonatal Hypoxic-Ischemic Brain Injury

doi: 10.3389/fneur.2021.607370

Figure Lengend Snippet: PLX3397 changes brain cytokine/chemokine profile in HI mice. (A) The total differentially expressed cytokines/chemokines in the brains of HI mice treated with PLX3397 or the vehicle. Brain tissues were collected at 48 h after HI induction and the protein levels were measured with a cytokine/chemokine profile ELISA array kit in which 111 factors were detected. n = 3 independent experiments. Each independent experiment needed 10 mice per group (5 males and 5 females). Unpaired two-tailed t -test. (B) The mRNA levels of representative differentially expressed factors related to regulation of chemotaxis (CCL2, CCL12), immune response (CD14, M-CSF, IL-7), and BBB integrity (ICAM-1, VEGF, and WISP-1). CX3CL1 was as a negative control that was not significantly changed in protein level between HI mice treated with PLX3397 or the vehicle (data not shown). Brain tissues were collected at 48 h after HI from HI mice treated with PLX3397 or the vehicle. n = 5 mice per group. Unpaired two-tailed t -test. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01.

Article Snippet: Cytokines in the cerebral tissue lysates were measured using a mouse XL cytokine array kit (ARY028, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Chemotaxis Assay, Negative Control

Journal:

Article Title: Intact Gram-Negative Helicobacter pylori , Helicobacter felis , and Helicobacter hepaticus Bacteria Activate Innate Immunity via Toll-Like Receptor 2 but Not Toll-Like Receptor 4

doi: 10.1128/IAI.72.11.6446-6454.2004

Figure Lengend Snippet: Murine macrophages are activated by whole H. pylori SS1 bacteria in a TLR2-dependent manner. (A) Wild-type mouse peritoneal exudate macrophages were stimulated with medium (left panel) or H. pylori SS1 bacteria (right panel). Following a 2-h incubation, RNA was extracted, labeled, and hybridized to a mouse SuperArray blot. The positions of inflammatory cytokine and cytokine receptor spots are indicated. (Bottom two rows) Negative controls included blanks and pUC18. Positive controls included the gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and housekeeping genes (those for cyclophilin A and actin). (B) Wild-type and TLR2 knockout (KO) mouse peritoneal exudate macrophages were stimulated with medium (None) or H. pylori SS1 bacteria (107 to 109 per well). Supernatants were harvested 18 h later, and IL-6 levels were determined by ELISA. (C) Wild-type, TLR2 knockout, or TLR4-deficient (def) ScN mouse peritoneal exudate macrophages were stimulated with medium alone or E. coli LPS (TLR4 ligand). Supernatants were harvested 18 h later, and IL-6 levels were determined by ELISA.

Article Snippet: Total mRNA (5 μg per blot) was reverse transcribed, labeled with biotin, and hybridized to a Mouse Inflammatory Cytokine SuperArray in accordance with the manufacturer's (SuperArray Inc., Frederick, Md.) instructions.

Techniques: Incubation, Labeling, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Association of Gamma Interferon and Interleukin-17 Production in Intestinal CD4 + T Cells with Protection against Rotavirus Shedding in Mice Intranasally Immunized with VP6 and the Adjuvant LT(R192G) ▿

doi: 10.1128/JVI.01877-06

Figure Lengend Snippet: Cytokine secretion after in vitro stimulation of lymphocytes isolated prior to challenge (time zero) from the MLN, PP, IEL, and LP of naive, VP6-immunized, LT(R192G)-immunized, and VP6/LT(R192G)-immunized mice. Lymphocytes isolated from 12 animals were pooled and placed in culture and stimulated with VP6 or left unstimulated for 18 h. Supernatants were harvested, and the cytokine concentrations were determined by ELISA. Values are expressed as the amount of cytokine secreted in the supernatants of the stimulated cultures minus the amount secreted in the unstimulated cultures (in pg/ml). The data are representative of one of three independent experiments.

Article Snippet: The GEArray Q series mouse common cytokine gene array (SuperArray Bioscience Corp., Frederick, MD) was used to determine mRNA expression of cytokines in all samples.

Techniques: In Vitro, Isolation, Enzyme-linked Immunosorbent Assay