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

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: Fig. 3 Pharmacological and genetic re-expression of TXNIP counteract 3T3-L1A CM effects on MCF-7 and MCF-7 TR BC cells. (A) Immunoblotting show ing 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) Im munoblotting 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