Journal: eBioMedicine
Article Title: Somatic structural variants drive upper tract urothelial carcinoma muscle invasiveness via activation of TPX2 transcription
doi: 10.1016/j.ebiom.2026.106182
Figure Lengend Snippet: An upstream structural variant interval enhances TPX2 expression and promotes invasive phenotypes in urothelial cancer cells. A. Schematic representation of somatic structural variants (SSVs) surrounding the TPX2 locus. A recurrent duplication event localised to the chr20:31000852–31001508 interval upstream of TPX2 was identified. This region contains eight tandem repeats of a 171-bp sequence. Two unique sgRNAs were designed to delete a 683-bp fragment spanning this interval. B. PCR and gel electrophoresis validation of CRISPR ribonucleoprotein (RNP)-mediated deletion of the TPX2 upstream interval in 5637 cells. The main band corresponding to the intact region (1025 bp) was markedly attenuated in TPX2-upstream knockout cells. (C, D). Quantitative RT-PCR (C) and Western blot (D) analyses showing that deletion of the chr20:31000852–31001508 interval significantly reduced TPX2 mRNA and protein expression in 5637 cells. (E, F) Quantitative RT-PCR (E) and Western blot (F) analyses demonstrating that transfection of a PCR-derived fragment corresponding to the TPX2 upstream interval increased TPX2 expression compared with control cells. G. Schematic of the dual-luciferase reporter construct in which the TPX2 promoter region (2331 bp, including exon 1) was cloned upstream of the Renilla luciferase (Rluc) gene in the psiCHECK-2 vector. H. Dual-luciferase reporter assay showing that co-transfection of the TPX2 promoter reporter with PCR-derived fragments from the chr20:31000852–31001508 interval significantly increased Rluc activity compared with co-transfection with a random 1-kb DNA fragment, indicating enhancer-like activity of this interval. (I, J) Western blot validation of TPX2 knockout (KO) (I) and TPX2 overexpression (OE) (J) in 5637 cells. (K, L) Wound-healing (K) and transwell migration and invasion assays (L) demonstrating that TPX2 knockout significantly impaired cell motility and invasive capacity. Scale bar = 1 mm in K, scale bar = 200 um in L. (M, N) Wound-healing (M) and transwell migration and invasion assays (N) showing that TPX2 overexpression markedly enhanced migratory and invasive properties of 5637 cells. Scale bar = 1 mm in M, scale bar = 200 um in N. Data are presented as mean ±SD (n = 3). Two-sided Mann-Whitney U test was used for comparisons between two groups. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). O. KEGG pathway enrichment analysis of genes downregulated upon TPX2 knockout (TPX2_KO vs. NC_KO). P. Gene set enrichment analysis (GSEA) of transcriptomic changes induced by amplification of the TPX2 upstream interval (Ups_OE vs. NC_OE). Pathways related to EMT, cell adhesion, and extracellular matrix remodelling were significantly enriched.
Article Snippet: The human bladder cancer-derived 5637 cells (ATCC) were cultured in RPMI 1640 culture medium supplemented with 10% foetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Invitrogen) at 37 °C with 5% CO2 and maximum humidity.
Techniques: Variant Assay, Expressing, Sequencing, Nucleic Acid Electrophoresis, Biomarker Discovery, CRISPR, Knock-Out, Quantitative RT-PCR, Western Blot, Transfection, Derivative Assay, Control, Luciferase, Construct, Clone Assay, Plasmid Preparation, Reporter Assay, Cotransfection, Activity Assay, Over Expression, Migration, MANN-WHITNEY, Amplification
ATCC is a verified supplier 
ATCC manufactures this product 
