FIGURE 5 from Identification of an Imidazopyridine-based Compound as an Oral Selective Estrogen Receptor Degrader for Breast Cancer Therapy
Year: 2023
Type: other
Abstract: X15695 reactivates p53. A, Western blot analysis of mortalin and p53 in immunoprecipitated extracts of MCF-7 cells treated with 1 μmol/L X15695 for 48 hours and lysed. 50 μg of cell extract was used as input control while 1,000 μg was immunoprecipitated with anti-p53 or IgG antibodies immobilized on protein A/G agarose beads. B, Western blot analysis of mortalin and BAG1 in immunoprecipitated extracts of MCF-7 cells treated with 1 μmol/L X15695 for 48 hours. MCF-7 cells were treated and processed as in A with the exception that the immunoprecipitation was carried out with a mouse anti-BAG1 antibody. C, Western blot analysis of BAG2 and BAG5 in immunoprecipitated extracts of T47D cells treated with 1 μmol/L X15695 for 48 hours. A total of 70 μg of cell extracts was used as input control while 1,000 μg was immunoprecipitated with anti-p53 or IgG antibodies immobilized on protein A/G agarose beads. D, Western blot analysis of p53 protein after transfection of MCF-7 and T47D cells with control and p53 siRNA and treatment with vehicle or X15695 (1 μmol/L) for 24 hours. β-actin was used for the loading control. E, Cell viability determination using MTT assay in control and p53 siRNA transfected cells treated with vehicle or X15695 (1 μmol/L) for 72 hours (n ≥ 3; ***, P ≤ 0.001; *, P ≤ 0.05). Cell-cycle analysis of MCF-7 (F) and T47D (G) cells treated with vehicle or 1 μmol/L X15695 for 48 hours. A total of 1 × 106 cells were stained with 7-AAD and used for flow cytometry measurement. Data were analyzed by FlowJo software. Apoptosis assessment of MCF-7 (H) and T47D (I) cells treated with 1 μmol/L X15695 for 48 hours. The cells were double stained with 7-AAD and Annexin V. Cells stained with 7-AAD or Annexin V alone were used for compensation of double staining. A total of 1 × 106 cells were used for the flow cytometry measurement. The quantified results are the mean ± SEM (n = 3; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001; ns is non-significant).
Authors: Mengwu Pan, Valeria Solozobova, Nane C. Kuznik, Nicole Jung, Simone Gräßle, Victor Gourain, Yvonne M. Heneka, Christina A. Cramer von Clausbruch, Olaf Fuhr, Ravi S. N. Munuganti, Danilo Maddalo, Christine Blattner, Antje Neeb, Adam Sharp, Laura Cato, Carsten Weiss, Rinath M. Jeselsohn, Veronique Orian-Rousseau, Stefan Bräse, Andrew C. B. Cato
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4385303183