Journal: Cell Communication and Signaling : CCS

Article Title: Adipocyte/Tumor cell crosstalk via IGF-1/TXNIP axis promotes malignancy and endocrine resistance in breast cancer

doi: 10.1186/s12964-025-02262-4

Figure Lengend Snippet: Effects of 3T3-L1A CM on TXNIP expression and activity in MCF-7 and MCF-7 TR cells. (A) Correlation heatmap of 27 selected genes of integrative MixOmics model. Tree-clustering was obtained according Ward’s method. 3T3-L1A CM and Control media (C) were shown in column annotation as red and blue, respectively. Similarly, the MCF-7 and MCF-7 TR cells were reported as green and black, respectively. ( B ) Horizontal bar plot visualizing the mean weight of each selected gene in the classification of a given condition in PLS-DA integrated model. The red and blue loadings represent the contribution of 3T3-L1A CM and C, respectively, as final outcome of interest. WLVs of LCs: weight of each loading vectors of latent components. (C) Enrichment analysis on terms from Biological Process Gene Ontology, KEGG, Reactome, and MSigDB annotating the 27 genes presenting similar modulation trends in both MCF-7 and MCF-7 TR cells treated with 3T3-L1A CM. The size and colour of each dot indicate number of genes per term and the BH-FDR value, respectively. The reference databases are: GO Biological Process (no star), Kegg ( * ) , MSigDB ( ** ) , and Reactome ( *** ) . Score: combined score of EnrichR. (D) Shortest Path Network (SPN) built by processing the 27 genes with similar deregulation trends in 3T3-L1A CM-treated MCF-7 and 3T3-L1A CM-treated MCF-7 TR cells. Edge colours and arrowheads indicate the type and the direction of protein interconnections (green arrow = positive effect; red arrow = negative effect; and grey arrow = unspecified interaction). (E) Real-time RT‐PCR for TXNIP mRNA levels in MCF-7 and MCF-7 TR cells treated (+) or not (-) for 48 h with 3T3-L1A CM. (F) Immunoblotting showing TXNIP protein expression in cells treated as indicated for 48 h. β-Actin was used as a control for equal loading and transfer. Numbers below the blots represent the mean of the band optical density expressed as fold over CM-untreated MCF-7 cells. (G) TXNIP promoter activity in cells treated as indicated for 24 h. (H) TRX activity in cells treated as indicated for 48 h. Mean ± S.E.M. *, P < 0.05; ***, P < 0.0005. (I) Spearman rank correlation between TXNIP and BMI in 103 BC patients with known BMI. (L) One-sided Mann-Whitney U test after back-transformation of log2 values for evaluating TXNIP expression in Normal Weight (NW, n = 131) and Obese (Ob, n = 141) patients. ( M ) Immunohistochemical detection of TXNIP expression in normal weight, overweight and obese patient-derived breast cancer tissues ( n = 65). Left Panel , statistically significant difference between TXNIP expression and BMI values was evaluated using the Mann-Whitney U test (*, P = 0.0382). Right Panel , representative IHC images showing low and high cytoplasmic TXNIP expression in breast cancer epithelial tissues. Pos. Tissue, IHC staining in tonsil, used as positive control tissue. Magnification: 20X. Scale bar: 50 μm

Article Snippet: The following day, cells were co-transfected with 500 ng of a firefly luciferase reporter plasmid containing the human TXNIP promoter region (Addgene plasmid #37084) and 50 ng of a Renilla luciferase control plasmid (pRL-TK, Promega) using Lipofectamine 3000(Thermo Fisher Scientific), according to the manufacturer’s recommendations.

Techniques: Expressing, Activity Assay, Control, Quantitative RT-PCR, Western Blot, MANN-WHITNEY, Transformation Assay, Immunohistochemical staining, Derivative Assay, Immunohistochemistry, Positive Control

Journal: Cell Communication and Signaling : CCS

Article Title: Adipocyte/Tumor cell crosstalk via IGF-1/TXNIP axis promotes malignancy and endocrine resistance in breast cancer

doi: 10.1186/s12964-025-02262-4

Figure Lengend Snippet: Pharmacological and genetic re-expression of TXNIP counteract 3T3-L1A CM effects on MCF-7 and MCF-7 TR BC cells. (A) Immunoblotting showing TXNIP protein expression in MCF-7 and MCF-7 TR cells treated (+) or not (-) with 3T3-L1A CM with/without the histone deacetylase inhibitor (HDAC) SAHA or the histone methyltransferase EZH2 inhibitor DZNeP for 48 h at 0.1, 1, 5, or 10 µM concentration. GAPDH was used as a control for equal loading and transfer. Soft agar growth (B) and Transmigration (C) assays in cells treated (+) or not (-) with 3T3-L1A CM ± SAHA (5 µM) or DZNeP (10 µM). (D) Immunoblotting showing TXNIP protein expression in cells stably overexpressing Myc-tagged human TXNIP protein (o.e.) or empty vector (c) using TXNIP ( upper panel ) and c-Myc ( lower panel ) antibodies. GAPDH was used as a control for equal loading and transfer. Soft agar growth (E) and Transmigration (F) assays in c and TXNIP o.e. cells treated (+) or not (-) with 3T3-L1A CM. Representative images of cell transwell migration are shown. Mean ± S.E.M. *, P < 0.05; **, P < 0.005; ***, P < 0.0005

Article Snippet: The following day, cells were co-transfected with 500 ng of a firefly luciferase reporter plasmid containing the human TXNIP promoter region (Addgene plasmid #37084) and 50 ng of a Renilla luciferase control plasmid (pRL-TK, Promega) using Lipofectamine 3000(Thermo Fisher Scientific), according to the manufacturer’s recommendations.

Techniques: Expressing, Western Blot, Histone Deacetylase Assay, Concentration Assay, Control, Transmigration Assay, Stable Transfection, Plasmid Preparation, Migration

Journal: Nature Biotechnology

Article Title: Systematic discovery of neoepitope–HLA pairs for neoantigens shared among patients and tumor types

doi: 10.1038/s41587-023-01945-y

Figure Lengend Snippet: a , Process overview for the cell engineering and MS analysis of peptides presented by polyantigen-expressing HLA monoallelic cell lines. KO, knockout. b , Vector map of the piggyBac polyantigen expression constructs used in this study. A single transcript containing 47 tandem neoantigens followed by seven control peptides and an IRES-linked mTagBFP2 (BFP) reporter is driven by an EF1a promoter. Neoantigens were either directly concatenated (no-linker) or interspersed by short flexible linker sequences (linker). c , Flow cytometric detection of HLA expression (W6/32 antibody-APC) and polyantigen cassette reporter (BFP) in selected cell lines or HLA knockout parental line. Here, ‘−’ indicates the absence of linkers and ‘+’ indicates presence of linkers in the polyantigen construct. d , Targeted immunopeptidomic detection of a previously described A*02:01-presented TP53 neoepitope (HMTEVVRHC, position 39) as well as two neoantigen control peptides from pp65 (NLVPMVATV, control 1) and IE-1 (VLEETSVML, control 4) known to be presented by the A*02:01 allele. a and b were created with BioRender.com .

Article Snippet: Unique HLA allele open reading frames (ORFs), each with a distinct 19-base pair (bp) DNA barcode, were cloned downstream of the human EF1a promoter (GenScript) in a custom-modified pLenti6.3 backbone (Thermo Fisher Scientific).

Techniques: Expressing, Knock-Out, Plasmid Preparation, Construct, Immunopeptidomics, Control