mouse il-2 is, research grade  (Miltenyi Biotec)

Journal: Advanced Science

Article Title: Coixenolide Enhances Antitumor Immunity of Cytotoxic CD8 + T Cells by Inhibiting S100A8/A9‐CD36 Axis in Obesity‐Associated Pancreatic Adenocarcinoma

doi: 10.1002/advs.202515759

Figure Lengend Snippet: Transcriptomic analyses revealed that coixenolide significantly inhibited S100a8/a9 + PAAD cells in HFD‐fed mice. (A) Subclusters of epithelial cells. (B) Malignancy score of epithelial cells. (C) The heterogeneity of epithelial cell clusters among different groups and tissue samples based on cell ratio. (D,E) Changes in the proportions of Cluster 2 (D) and Cluster 8 (E) tumor cells. n = 7 for HFD group and n = 6 for CD group. Data are presented as mean ± SD; a two‐sided t ‐test was used. (F) The upregulated genes across 21 tumor cell subsets. (G) Pathway enrichment associated with the top 100 upregulated genes (ranked by p ‐value) of Clusters 2 and 8 tumor cells. (H) CytoTRACE score and distribution of tumor cell clusters. (I) Shared genes of Clusters 2 and 8. (J) The colocalization of S100A8/A9 (yellow), Pan‐CK (red), and DAPI (deep blue) in the tumor tissues of CD‐ and HFD‐fed mice; representative of three independent experiments. (K) The expression patterns of S100A8 and S100A9 between tumor and normal pancreatic tissues using the GEPIA webtool; n = 179 (tumor tissue) and 171 (normal tissue); a two‐sided Wilcoxon test was used. (L) The colocalization of S100A8/A9 (yellow), insulin (pink), Pan‐CK (white), and DAPI (deep blue) in the peritumoral pancreatic tissue, as well as in tumor tissues in PAAD patients. (M,N) Kaplan–Meier survival plot indicating that patients with high levels of S100A8 (M) or S100A9 (N) have a worse clinical outcome in the TCGA PAAD cohort; log‐rank test was used. (O,P) The expressions of S100A8 (O) and S100A9 (P) between patients with PAAD of different invasion levels, grades, and AJCC stages. The data were sourced from the TCGA cohort; a two‐sided t ‐test was used. (Q) Expression levels of S100a8 and S100a9 in Clusters 2 and 8 tumors of HFD‐fed mice following coixenolide treatment. A two‐sided t ‐test was used. (R,S) Western blot (R) and flow cytometry plot (S) measuring S100a8/a9 expression in Pan02 cells following coixenolide treatment. n = 4 independent experiments; two‐sided t ‐test was used. (T) The levels of S100a8/a9 in the supernatants of Pan02 cells with or without coixenolide treatment were determined using ELISA. n = 3 independent experiments; two‐sided t ‐test was used. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CD8 + T cell supernatants were collected, and the secretion of TNF‐α (Mouse TNF‐alpha ELISA Kit, RK00027, Abclonal), IFN‐γ (Mouse IFN‐gamma ELISA Kit, RK00019, Abclonal), and GZMB (Mouse Granzymes B ELISA Kit, RK00007, Abclonal) was quantified using specific ELISA kits following the standard protocols.

Techniques: Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Coixenolide Enhances Antitumor Immunity of Cytotoxic CD8 + T Cells by Inhibiting S100A8/A9‐CD36 Axis in Obesity‐Associated Pancreatic Adenocarcinoma

doi: 10.1002/advs.202515759

Figure Lengend Snippet: Cell–cell communication analysis indicated that S100a8/a9 served as a critical target for coixenolide, regulating the interactions between PAAD cells and CD8 + T cells. (A) The overview of cell–cell interactions between malignant epithelial cells and T cells in the tumor tissues treated with vehicle control and coixenolide, respectively. (B) The intensity of intercellular signaling, showing both the sent and received signals. The cells in the upper right corner exhibit the strongest incoming and outgoing interactions. S100a8/a9 + tumor cell clusters (Clusters 2 and 8) are highlighted in red boxes. Cytotoxic CD8 + T and Treg cells are indicated by red arrows. (C) The outgoing communication patterns of epithelial cells and T cells in the tumor of HFD‐fed mice with or without coixenolide treatment. (D) CD8 + T cell killing assays were conducted using coculture of Pan02 and CD8 + T cells. Pan02 or CD8 + T cells were pretreated with 25 mg/mL coixenolide before coculture. (E) Primary CD8 + T cells were activated by incubation with CD3/CD28 antibody and labeled with carboxy‐fluorescein succinimidyl ester (CFSE). (F–H) The levels of TNF‐α (F), IFN‐γ (G), and Granzyme B (GZMB) (H) in the supernatants after co‐culture of CD8 + T and Pan02 cells pretreated with coixenolide (+) or vehicle control (−) were determined using ELISA. (I–K) The expression of TNF‐α (I), IFN‐γ (J), and Granzyme B (GZMB) (K) in CD8 + T cells after co‐culture of CD8 + T cells and Pan02 cells pretreated with coixenolide (+) or vehicle control (−) was determined using flow cytometry. (L) Flow cytometry analyses of T cell exhaustion markers (CTLA‐4) on CD8 + T cells after coculture of Pan02 and CD8 + T cells pretreated with coixenolide (+) or vehicle control (−). (M,N) Flow cytometry analyses of CD8 + T cell killing (M) and division (N) activities. CD8 + T cells were cocultured with S100a9 knockout (KO) or wild‐type (WT) Pan02 cells. (O–Q) The levels of TNF‐α (O), IFN‐γ (P), and GZMB (Q) in the supernatants after coculture of CD8 + T cells with WT or S100a9 KO Pan02 cells were determined using ELISA. n = 3 or 4 independent coculture experiments or replicates. One‐way ANOVA with Tukey's post‐hoc test (E–L), two‐sided t ‐test (M–N), or two‐way ANOVA (O‐Q) was used. All p values are two‐sided. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CD8 + T cell supernatants were collected, and the secretion of TNF‐α (Mouse TNF‐alpha ELISA Kit, RK00027, Abclonal), IFN‐γ (Mouse IFN‐gamma ELISA Kit, RK00019, Abclonal), and GZMB (Mouse Granzymes B ELISA Kit, RK00007, Abclonal) was quantified using specific ELISA kits following the standard protocols.

Techniques: Control, Incubation, Labeling, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Knock-Out