coding sequences  (Twist Bioscience)

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

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: Donor mito-mTagBFP2 can be free from endocytic vesicles. HUVECs expressing cell surface GFP and endosome-targeted Rab5a-TagRFP (magenta) transplanted with mito-mTagBFP2 displaying anti-GFP nanobody (cyan). The videos were recorded 6 h and 1 day after mitochondrial transplantation, respectively. Two different cells are shown. Scale bar, 5 mm.

Article Snippet: For TagBFP2 targeting into the matrix, the mTagBFP2 coding DNA sequence was fused to COX8 matrix-targeting signal peptide, synthesized by Twist Biosciences, and inserted into a pCMV backbone.

Techniques:

Journal: Nature

Article Title: Cell-type-targeted mitochondrial transplantation rescues cell degeneration

doi: 10.1038/s41586-026-10391-0

Figure Lengend Snippet: a. Top, schematic diagram of the construct used for directing a nanobody to the outer membrane of mitochondria. Bottom, a super-resolution image of an HEK293T cell with mitochondria-targeted nanobody detected by anti-alpacaV H H antibodies (magenta). Cell nuclei are labelled with Hoechst (blue). Mitochondria matrix is labelled with dsRed2 (cyan). 3D SIM, three-dimensional structural illumination microscopy. The construct was validated in at least three independent experiments. b. Top, schematic diagram of the construct used for directing GFP to the cell surface. Bottom, a super-resolution image of an HEK293T cell with cell surface-targeted GFP detected by anti-GFP antibodies (green). Cell nuclei are labelled with Hoechst (blue). The construct was validated in at least three independent experiments. c. HEK239T cells transplanted with donor mitochondria displaying the anti-GFP nanobody, two hours after transplantation. HEK293T cells are transfected with cell-surface mCherry (cyan) or GFP (green). Nanobodies are detected by anti-alpacaV H H antibodies (magenta). d. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria two hours after transplantation. n = 6, P < 0.0001 (top) and P = 0.0012 (bottom), two-sided Welch’s t test. e. HEK239T cells transplanted with donor mitochondria displaying the anti-mCherry nanobody, two hours after transplantation. HEK293T cells were transfected with cell-surface GFP (green) or mCherry (cyan). Nanobodies detected by anti-alpacaV H H antibodies (magenta). f. Quantification of the efficacy of the delivery of nanobody-displaying mitochondria, two hours after transplantation. n = 8, P = 0.0003 (top) and P = 0.0012 (bottom), two-sided Welch’s test. g. Live-imaged endothelial cells expressing cell surface GFP and endosome-targeted RAB5A-TagRFP with (top) and without (bottom) transplanted with mito-mTagBFP2 (cyan) displaying anti-GFP nanobody, six hours after mitochondrial transplantation. White arrows, endosome-free donor mitochondria (confirmed in at least three independent experiments). For an example of mito-mTagBP2 in endothelial cells transplanted without the anti-GFP nanobody, see Supplementary Fig. . h. Donor mito-mTagBFP2 (cyan) inside endocytic vesicles (red arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. i. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta) (from g), six hours after mitochondrial transplantation. j. Donor mito-mTagBFP2 (cyan) free from endocytic vesicles (white arrows) labelled with RAB5A-TagRFP (magenta), 24 h after mitochondrial transplantation. Outlined region, mito-mTagBFP2 free from endocytic vesicles and lysosomes (yellow). Lysosomes are stained with LysoTracker Deep Red dye. The experiment was repeated at least three times with similar results. k. Quantification of proportion of endosome-free mitochondria by pixel-based co-localization analysis. Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P = 0.0756, two-sided Mann-Whitney U test. l. Quantification of abundance of endosome-free mitochondria by pixel-based co-localization analysis. The values were normalized to cell size (µm 2 of donor mitochondria area per 1 µm 2 cell area). Mito-mTagBFP2: n = 30; mito-mTagBFP2 + anti-GFP nanobody: n = 52, P < 0.0001, two-sided Mann-Whitney U test. m. Endothelial cells stained with pH-dependent lysosome staining dye pHLys Red (yellow). Cells expressed cell surface GFP and were transplanted with mito-mTagBFP2 displaying anti-GFP nanobody or no binder. In addition, Bafilomycin A1 was used as a positive control for pH acidification change in lysosomes. For mitochondria transplanted conditions, images of cells positive for mito-mTagBFP2 are shown (Supplementary Fig. ). n. Quantification of pH changes in lysosomes relative to untreated condition. Untreated: n = 6; Bafilomycin A1: n = 4; mito-mTagBFP2: n = 4; mito-mTagBFP2 + anti-GFP nanobody: n = 6, Untreated vs. Bafilomycin A1: P = 0.0036, Untreated vs. mito-mTagBFP2: P = 0.8235, Untreated vs. mito-mTagBFP2 + anti-GFP nanobody: P = 0.9648, Welch’s ANOVA test corrected with two-sided Dunnett’s test for multiple comparisons. o. Live-imaged endothelial cell expressing cell surface GFP (green), and transplanted with mito-dsRed2 (cyan) displaying anti-GFP nanobody, four days after mitochondrial transplantation. The cell is outlined with a grey dashed line. The zoomed-in region is outlined with a white dashed square. Two timeframes are shown on the right. The tracked mitochondrion is indicated with a red arrow. The experiment was repeated at least three times with similar results. p. Live-imaged endothelial cell expressing cell surface GFP and transplanted with mito-dsRed2 (cyan) displaying outer membrane anti-GFP nanobody, four days after mitochondrial transplantation. Mitochondria are labelled with 50 nM MitoTracker Deep Red (magenta). The zoomed-in region is outlined with a white dashed square and the tracked mitochondrion is indicated with a white arrow. The experiment was repeated at least three times with similar results. q. Labelling of donor and native mitochondria with MitoTracker Deep Red dye in live-recorded endothelial cells. At the used concentration, the dye stained both native (black) and donor mitochondria (cyan) with stronger enrichment in the native mitochondria. Donor mitochondria positive for matrix-labelled dsRed2 and MitoTracker Deep Red are indicated with red arrows. NS not significant, ** P < 0.01, *** P < 0.001. Data, mean ± s.e.m and median for k, l. Scale bars, 2.5 µm (a, b), 25 µm (c, e), 5 µm (g, h, i, o, p, q), 10 µm (j), 20 µm (m).

Article Snippet: For TagBFP2 targeting into the matrix, the mTagBFP2 coding DNA sequence was fused to COX8 matrix-targeting signal peptide, synthesized by Twist Biosciences, and inserted into a pCMV backbone.

Techniques: Construct, Membrane, Microscopy, Transplantation Assay, Transfection, Expressing, Staining, MANN-WHITNEY, Positive Control, Concentration Assay