dsred coding sequence  (Promega)

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 is positively correlated with favorable prognosis in breast cancer. ( a ) High TRAF3 mRNA expression levels are associated with better OS (Living vs. Diseased, Mann–Whitney U test), lower disease stage (Bonferroni correction), lower lymph node stage (N) (N0 vs. N1: p = 0.004, Bonferroni correction) and lower tumor stage (T) (T1 vs. T3: p = 0.019, Bonferroni correction) in the TCGA-BRCA cohort. ( b ) High TRAF3 mRNA expression presents with a statistically significant better OS ( p = 0.00405) and DMFS ( p = 0.00729) in the ER-negative breast cancer cohort employed by GOBO, with ER-positive disease presenting a similar association despite not reaching statistical significance.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Forced TRAF3 expression in breast cancer cell lines induces partial EMT and affects cell proliferation. ( a ) Invasion, migration and colony formation assays depicting an opposing phenotype between migratory and proliferative states of MCF7-TRAF3 cells. ( b ) Western blot analyses for the indicated proteins in MDA-MB-231 and MCF-7 cells (control and TRAF3 expressing). ( c ) ICC for the indicated proteins in MCF-7 cells, indicating significant downregulation of key molecules upon TRAF3 expression (ns: no significance; *** p < 0.001 Student’s t -test).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Migration, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Identification of interactors, pathways and processes of TRAF3 in breast cancer. ( a ) Volcano plot of significant TRAF3 interactions in MCF-7 cells (FDR < 0.05). ( b ) Top 20 enriched pathways (Metascape) among proteins that interact with TRAF3 in MCF-7 cells with −log10(Padj) > 10 −20 . ( c ) Significantly enriched pathways among genes co-expressed with TRAF3 in the TCGA BRCA cohort. ( d ) Representative BRCA cases from the TCGA cohort presenting with High and Low TILs (upper panel). High TRAF3 mRNA expression is correlated ( p = 0.02, Mann–Whitney U Test) with High stromal TILs in the TCGA BRCA cohort ( n = 200).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression across cell populations in the scRNA human breast cancer dataset. ( a ) UMAP visualization of 81,389 quality-filtered single cells derived from the Breast Cancer Atlas, colored by cell type annotation. ( b ) Feature plot showing log-normalized TRAF3 expression projected onto the UMAP embedding. ( c ) Violin plots depicting log-normalized TRAF3 expression across each of the cell types. Statistical comparisons were performed using Wilcoxon rank-sum tests, comparing each cell type against all remaining cells, followed by Benjamini–Hochberg correction for multiple testing. Asterisks (*) indicate adj p -values < 0.05. ( d ) Volcano Plot of Differential expression of TRAF3 -positive ( TRAF3 +) vs. negative ( TRAF3 -) Cancer Epithelial (CE) cells. The x-axis represents the log 2 fold change of expression in TRAF3 -positive versus TRAF3 -negative cells, and the y-axis shows the −log 10 adjusted p -value (FDR). Points are colored according to FDR significance, while labels highlight specific immunologically relevant genes, colored according to the following categories: (i) Immunogenicity—Immunogenicity/Antigen Presentation; (ii) MHC-I—MHC class I pathway (CD8 + T-cell recognition); (iii) MHC-II—MHC class II (tumor-intrinsic or antigen-presenting cell mediated); (iv) Checkpoint—Checkpoint blockade/Immune Modulation; (v) Infiltration—Increase immune infiltration into tumors; and (vi) Non-self—Promote tumor cell recognition as “non-self”. Selected genes of interest not in the above categories are colored black (‘Other’ category). ( e ) Gene Ontology (GO) Enrichment Analysis of the filtered top DE genes (FDR < 0.05 & |log2FC| > 0.1) identified via differential expression analysis between TRAF3 + and TRAF3 -cancer epithelial (CE) cells. X-axis represents the Fold Enrichment, and y-axis represents the immune-related Biological Process and Molecular Function GO terms, grouped into clusters based on functional similarity (for the full GO term graph with all the immune and non-immune related GO terms, see ). Dot size is analogous to the number of specific genes associated with each GO term, while their color gradient corresponds to the FDR-adjusted p -value (Q value). Abbreviations used include the following: CE (Cancer Epithelial cells), NE (Normal Epithelial cells), PVL (PeriVascular-Like cells), CAFs (Cancer-Associated Fibroblasts), PR (Positive Regulation), R (Regulation), prd (production), MM (Molecular Mediator), MBP (Macromolecule Biosynthetic Process), MMP (Macromolecule Metabolic Process), CR (Cellular Response), env/tal (environmental), RSP (receptor signaling pathway), SP (signaling pathway), resp. (response), ext. (external), and If-M (interferon-mediated).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Immunopeptidomics, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression in cancer cells affects PBMC subpopulations and cytokine expression. ( a ) FACs analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the downregulation of the CD25+CD127low (Tregs) subpopulation of CD4+ T cells. ( b ) FACS analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the upregulation of the CD56+CD16- subpopulation of NK-cells. ( c ) Diagrams depicting absolute quantification of IFN-γ, TNF-α and IL-10 in the supernatants of co-cultured PBMCs/MCF7-TRAF3 cells. ( d ) FACs analysis for live/dead MCF-7 breast cancer cells co-cultured with PBMCs depicting a shift from alive to dead cells in the MCF7-TRAF3 cell population in comparison to MCF7-control cells. ( e ) IHC stain for PD-L1 (CD274) on MCF7-control and MCF7-TRAF3. Arrowheads depict PD-L1 expression only on MCF7-control cells. ( f ) Schematic illustration of a proposed model of TRAF3 action in breast cancer epithelial cells and on the surrounding tumor microenvironmental cells.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Cell Culture, Quantitative Proteomics, Comparison, Control, Staining