Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: Identification of the target of sTN58 aptamer on TNBC cell surface. (A) Schematic representation of biotin-sTN58-mediated affinity purification. Membrane-protein fraction from Cis-Pt-R cells were subjected to a preclearing step to remove non-specific components prior to the sTN58-mediated precipitation. The colloidal Blue-stained SDS-PAGE (10 %) displayed is utilized for the analysis of target purification mediated by the sTN58 aptamer. The molecular weights of protein markers are reported. Lane 1, molecular markers; lane 2, membrane extracts; lane 3, 15 μg aliquot of unbound proteins from SCR-mediated purification; lane 4, proteins captured with sTN58. Red boxes indicate the regions excised for MS analyses. (B) Comparison of transcript expression values of best candidates in different BC cell lines. The normalized transcript expression values (nTPM), according to HPA, are reported relative to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. Box indicates the 5 candidates chosen for experimental validation. (C) Immunoblot analysis of EphA2, CD44, integrin β1, myoferlin, liprin β1 and ZO-1, and of the housekeeping proteins α-tubulin and vinculin. The molecular weights of protein markers are reported. Black dashed lines delineate the boundary between non-contiguous lanes of the same gel. (D) The histogram shows the relative fold-change in expression levels of the indicated proteins compared to the housekeeping protein α-tubulin or vinculin, normalized to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. (E) Binding affinity (1/Kd) of TN58 aptamer to the indicated cell lines expressed relative to MDA-MB-231 target cells. Dose response curves and binding affinity calculations for MDA-MB-231 and Cis-Pt-R and Dox-R chemoresistant cells, as well as non-TNBC BT-474, MCF-7 and A431 cells were previously reported [ , ]. The dose response curve used for Kd calculation in relation to BT-549 is shown in . "NB", no binding.

Article Snippet: Filters were incubated overnight at 4 °C with the following primary antibodies: CD44 (lysates from human cell lines), Zonula occludens-1 (ZO-1, D7D12), platelet-derived growth factor receptor β (PDGFRβ, 28E1), phospho-44/42 MAPK (extracellular signal-regulated kinase 1/2, ERK1/2, D13.14.4E, indicated as p-ERK1/2), phospho-Akt (Ser473, indicated as p-Akt), Akt, Met (25H2), vimentin (D21H3), E-Cadherin (24E10), vinculin (E1E9V), α-tubulin (DM1A) (Cell Signaling Technology Inc., Danvers, MA, USA); CD44 (lysates from murine 4T1 cells, ab157107), integrin β1 (ITGB1, ab179471) (Abcam, Cambridge, UK); liprin β1, Ephrin Type-A Receptor 2 (EphA2), ERK1 (C-16) (Santa Cruz Biotechnology, Santa Cruz, CA); myoferlin (MYOF, HPA014245, Sigma-Aldrich) and programmed cell death-ligand 1 (PD-L1)/CD274 (Proteintech Group, Inc.).

Techniques: Affinity Purification, Membrane, Staining, SDS Page, Purification, Comparison, Expressing, Biomarker Discovery, Western Blot, Binding Assay