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: For dual immunofluorescence staining of CD44 and integrin β1, 8.0 × 10 4 Cis-Pt-R or A431 cells, were fixed and blocked as above, and incubated with CD44-PE (IM7, Santa Cruz Biotechnology) and integrin β1 (R&D system) antibodies, as above.

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

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: sTN58 aptamer and CD44 Ab colocalize with integrin β1 Ab on Cis-Pt-R cells. (A) Following 5 min incubation at RT with 2 μM Alexa 647-sTN58, Cis-Pt-R cells were stained with integrin β1 Ab, visualized by confocal microscopy, and photographed. (B) Cell lysates from Cis-Pt-R cells left untreated or treated for 48 h with 100 nM siRNA ctrl or si-ITGB1 were analyzed by immunoblotting with integrin β1, CD44 and anti-α-tubulin antibodies. Molecular weights of protein markers are reported. (C) The histogram shows the protein expression/α-tubulin ratio based on the densitometric signals. Values are shown relative to untreated samples, arbitrarily set to 1. (D) Binding of integrin β1-APC-Cy7 Ab ( left ) and Alexa 647-sTN58 ( right ) to Cis-Pt-R cells following 48 h transfection with siRNA ctrl (gray) and si-ITGB1 (pink). (E) The histogram shows gMFI of si-ITGB1-transfected cells treated with Alexa 647-sTN58 or integrin β1-APC-Cy7 Ab, normalized to the gMFI of untreated cells, and expressed as percentage with respect to siRNA ctrl-transfected cells. Bars depict mean ± SD of two independent experiments. ∗∗∗ P < 0.001; ns, no significant. (F) Confocal microscopy analyses of A431 cells treated with sTN58 and stained with integrin β1 Ab, as in A, or stained with CD44-PE and integrin β1 antibodies. Alexa 647-SCR was used as a negative control. In A, F, aptamers and CD44-PE Ab are visualized in red, integrin β1 Ab in green and nuclei in blue. All digital images were captured at the same setting to allow direct comparison of staining patterns. Magnification 63×, 1.0× digital zoom, scale bar = 10 μm. Co-localization results appear yellow in the merged images. Arrowheads indicate some co-localization points between sTN58 and integrin β1 Ab (Overlap Coefficient, 0.80). (G) Flow cytometry analyses of A431 cells treated with CD44-PE Ab, Alexa 647-sTN58 and integrin β1-APC-Cy7 Ab. (H) Quantification of the gMFI of sTN58-, CD44 PE- and integrin β1-APC-Cy7-treated cells normalized to the gMFI of the untreated cells. Bars depict mean ± SD of three independent experiments. ∗∗∗∗ P < 0.0001 relative to untreated cells; ns, no significant.

Article Snippet: For dual immunofluorescence staining of CD44 and integrin β1, 8.0 × 10 4 Cis-Pt-R or A431 cells, were fixed and blocked as above, and incubated with CD44-PE (IM7, Santa Cruz Biotechnology) and integrin β1 (R&D system) antibodies, as above.

Techniques: Incubation, Staining, Confocal Microscopy, Western Blot, Expressing, Binding Assay, Transfection, Negative Control, Comparison, Flow Cytometry

Journal: Biomedicines

Article Title: Angiogenesis in the Outer Membrane of Chronic Subdural Hematomas through Thrombin-Cleaved Osteopontin and the Integrin α9 and Integrin β1 Signaling Pathways

doi: 10.3390/biomedicines11051440

Figure Lengend Snippet: Western blots showing the expression of integrin α9β1 and the subsequent angiogenic pathway molecules in the outer membrane of chronic subdural hematomas from eight patients. Integrins β1 and α9, vinculin, talin-1, focal adhesion kinase (FAK), FAK phosphorylated at Tyr397 (p-FAK at Tyr397), paxillin, α-actinin and Src were detected in almost all cases. Positive controls are shown in the right lanes and suggest that these molecules were correctly detected. RAW 264.7, murine leukemia macrophage cell line lysate; rat liver, rat liver whole cell lysate; A431 cell lysate, epidermoid carcinoma cell lysate; rat brain lysate, rat brain whole cell lysate.

Article Snippet: Positive controls were RAW264.7 cell lysate (Cell Signaling Technology), rat liver lysate (BD Bioscience, Franklin Lakes, NJ, USA), A431 cell lysate (Santa Cruz Biotechnology, Dallas, TX, USA) and rat brain lysate (BD Bioscience).

Techniques: Western Blot, Expressing, Membrane