Journal: Frontiers in Oncology

Article Title: Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes

doi: 10.3389/fonc.2023.1249895

Figure Lengend Snippet: Generation and RNA-sequencing of epithelial and mesenchymal (post-EMT) subpopulations of mammary epithelial cell-derived EMT models. (A) EpCAM-positive and EpCAM-negative subpopulations of HMLE cells were separated using anti-EpCAM-conjugated capture beads. The cell fractions were flow cytometry-sorted into an epithelial (CD24 High /CD44 Low /EpCAM High ) and a mesenchymal (CD24 Low /CD44 High /EpCAM Low ) population. (B) MCF10A cells were left untreated or treated with 10 ng/ml TGF-β for 8 days. (C) D492 and D492M were cultured in 2D monolayers. (D) Flow chart of the RNA-Sequencing experiments. (E) Heatmap showing the fold change (post-EMT versus epithelial) of selected EMT marker genes, EMT transcription factors, and housekeeping genes. (F) Positively enriched Hallmarks with the post-EMT cells identified by Gene Set Enrichment Analysis (GSEA). The data from the three cell models are analyzed separately or combined (right panel). Ep, Epithelial; Mes, Mesenchymal; TFs, Transcription factors.

Article Snippet: To induce EMT, MCF10A cells were treated with 10 ng/ml recombinant human TGF-β for 8 days (Bio-Techne, CFQ-331134).

Techniques: RNA Sequencing Assay, Derivative Assay, Flow Cytometry, Cell Culture, Marker

Journal: Frontiers in Oncology

Article Title: Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes

doi: 10.3389/fonc.2023.1249895

Figure Lengend Snippet: ZEB1 acts as a transcriptional activator and is required for acquisition of post-EMT gene expression pattern. (A, B) SNAI1 , TWIST1 , ZEB1 , and ZEB2 expression in basal-like patients from the TCGA (A) post-EMT cluster 1 (C1) and partial-EMT cluster 5 (C5) and METABRIC post-EMT cluster 1 (C1) and partial-EMT cluster 3 (C3). Expression values are log2(RPKM+1). (C) and (D) Correlation analysis for individual genes withing the 265-gene mammary EMT signature with ZEB1 and SNAI1 in TCGA (C) and METABRIC (D) . (E) Hierarchical clustering of the mammary EMT signature in wild type and ZEB1 knockout MCF10A cells left untreated of treated with TGF-β. Genes found to be up- or downregulated in the EMT cell lines models (Up/Down), gene clusters from the breast cancer cell lines (GC-CL) clustering, and gene clusters from the TCGA (GC-TCGA) clustering are shown as side banners on the left side of the heatmap. Expression values are row-based z-normalized counts per million (CPM). (F) Hierarchical clustering of the mammary EMT signature in HMLER ZEB1 knock out cells (ZEB1 ko), ZEB1 ko cells that overexpress SNAI1 (SNAI1 oe), and SNAI1-expressing cells that are rescued by ectopic expression of ZEB1 (ZEB1 rescue). * P<0.05; *** P<0.001; **** P<0.0001. ns, not significant.

Article Snippet: To induce EMT, MCF10A cells were treated with 10 ng/ml recombinant human TGF-β for 8 days (Bio-Techne, CFQ-331134).

Techniques: Expressing, Knock-Out

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Notch Ligand Delta-Like 4 Blockade Alleviates Experimental Autoimmune Encephalomyelitis by Promoting Regulatory T Cell Development

doi: 10.4049/jimmunol.1100725

Figure Lengend Snippet: Dll4-mediated signaling regulates Treg expansion in vivo and in vitro. EAE was induced in C57BL/6 mice immunized with MOG(35–55)/CFA and treated with anti-DLL4 blocking Ab or control IgG every other day and for a total of five injections (arrows) starting on the day of the immunization. A, Flow cytometry plots from individual mice from each group for CD4 and Foxp3 staining of splenocytes or lymph node cells on DPI 10 (preclinical disease) and spinal cord cells on DPI 14 (peak of disease). Results are representative of three independent experiments with five mice per group. B, Statistical analysis of staining described in A. *p < 0.05, **p < 0.005 by unpaired t test. CD4+Foxp3− T cells (C) or total CD4+ T cells (D) were isolated from naive Foxp3.GFP.KI mice and stimulated in vitro with plate-bound anti-CD3 and anti-CD28 (1 µg/ml) in the presence of plate-bound rDll4 or control IgG (2 ug/ml). Optimal dose TGF-β (3 ng/ml) or IL-2 (20 ng/ml) was added to the culture medium when indicated and then supplemented to the culture medium on day 2 of culture. Cells were washed and stained for CD4 and Foxp3 on day 4 of culture. Results are representative of three independent experiments.

Article Snippet: Recombinant mouse Dll4-Fc fusion protein, IL-2, and TGF-β1 were purchased from R&D Systems.

Techniques: In Vivo, In Vitro, Blocking Assay, Flow Cytometry, Staining, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Allergen-responsive CD4 + CD25 + Regulatory T Cells in Children who Have Outgrown Cow's Milk Allergy

doi: 10.1084/jem.20032121

Figure Lengend Snippet: Generation of the immunosuppressive cytokines TGF-β1-LAP and IL-10 by PBMCs purified from blood of allergic or tolerant children before and 1 wk after in vivo challenge with cow's milk. (A) Production of TGF-β1-LAP determined in supernatants from PBMCs stimulated for 96 h with β-LG (1 mg/ml) or PHA (50 μg/ml) by subtracting the TGF-β1-LAP concentration in unstimulated wells. (B) Production of IL-10 under the same culture conditions as in A. Student's t test was used for statistical analysis.

Article Snippet: An ELISA was performed in round-bottom wells coated overnight with a pretitrated optimal concentration of mAb to human TGF-β in its inactive complex (anti–TGF-β1-LAP; 2 μg/ml, clone 27235.1; RD System Europe Ltd.).

Techniques: Purification, In Vivo, Concentration Assay