FIGURE 4 from Identification of an Imidazopyridine-based Compound as an Oral Selective Estrogen Receptor Degrader for Breast Cancer Therapy
Year: 2023
Type: other
Abstract: X15695 stabilizes p53. Western blot analysis of p53 in extracts of MCF-7 (A) and T47D (B) cells treated with vehicle or the indicated concentrations of X15695 for 48 hours. Anti-β-actin antibody was used for the loading control (same as it appears in Fig. 3A and B). Quantitative RT-PCR of p53 expression in MCF-7 (C) and T47D (D) cells after treatment with vehicle (DMSO) or the indicated concentrations of X15695 for 48 hours. The results are the mean ± SEM (n = 3; ns, nonsignificant). E, Western blot analysis of p53 in extracts of MCF-7 cells treated with 0.5 μmol/L MG132 in the presence and absence of 1 μmol/L X15695 for 48 hours. Anti-β-actin antibody was used for the loading control. F, Quantification of the p53 protein band intensities relative to the intensity of the β-actin signal. The results are the means ± SEM (n = 3; *, P ≤ 0.05; ns, nonsignificant result). Western blot analysis of MDM2 levels in extracts of MCF-7 (G) and T47D (H) cells after treatment with the indicated concentration of X15695 for 48 hours. Anti-β-actin antibody was used for determination of equal protein level. The protein band intensities were quantified and presented below. Measurement of DCF fluorescence in MCF-7 (I) and T47D (J) cells treated with the indicated concentrations of X15695 or DMSO for 48 hours. The signals were normalized to DNA content of the samples measured after Hoechst 33258 staining. The results are the mean ± SEM (n = 3; ***, P ≤ 0.001; ****, P ≤ 0.0001). K, Reduced H2DCF-DA oxidation assay showing the effect of NAC on ROS production in MCF-7 cells treated with X15695 for 48 hours. Results represent the mean H2DCF-DA oxidation normalized to DNA content (n = 1 with 6 technical replicates). L, Western blots of lysates of MCF-7 cells treated with the indicated concentrations of NAC 1 hour prior to treatment with 1 μmol/L X15695 or DMSO for 48 hours. Expression of p53 was monitored using anti-p53 antibody and anti-β-actin antibody was used as a protein loading control. M and N, Immunofluorescence images showing p53 localization in MCF-7 and T47D cells treated with vehicle (DMSO) and 1 μmol/L X15695 for the indicated time periods. For staining, anti-p53 antibody was used followed by goat anti-mouse IgG, Alexa Fluor 488 antibody – green) and DAPI (magenta; M) or Alexa Fluor 488 antibody – red) and DAPI (light blue; N).
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: W4385303137