FIGURE 6 from Identification of an Imidazopyridine-based Compound as an Oral Selective Estrogen Receptor Degrader for Breast Cancer Therapy
Authors: Pan Mengwu , Solozobova Valeria , Kuznik Nane C. , Jung Nicole , Gräßle Simone , Gourain Victor , Heneka Yvonne M. , Cramer von Clausbruch Christina A. , Fuhr Olaf , Munuganti Ravi S. N. , Maddalo Danilo , Blattner Christine , Neeb Antje , Sharp Adam , Cato Laura , Weiss Carsten , Jeselsohn Rinath M. , Orian-Rousseau Veronique , Bräse Stefan , Cato Andrew C. B. 
Published: July 2023

<p>X15695 and fulvestrant downregulate ERα level and proliferation of breast cancer cell proliferation <b>A,</b> Western blot analysis of ERα in extracts of MCF-7 cells treated for the indicated timepoints with 1 μmol/L of fulvestrant or X15695 or DMSO (vehicle). β-actin was used for the loading control. The protein signals were quantified and presented in percentages as the signal of ERα relative to the β-actin signal (right). The value at zero time of treatment was set to 100%. The values are the mean ± SEM (<i>n</i> = 3; *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.01; ***, <i>P</i> ≤ 0.001). <b>B,</b> Western blot analysis of ERα in extracts of MCF-7 cells treated with the indicated concentration of fulvestrant or X15695 for 8 hours. β-actin was used for the loading control. The protein signals were quantified and presented in percentages relative to the vehicle-treated sample (right). The values are the mean ± SEM (<i>n</i> = 3; ****, <i>P</i> ≤ 0.0001). <b>C,</b> Measurement of cell viability by MTT assay of tamoxifen resistant cells (TRMCF-7) treated with the indicated concentrations of X15695, fulvestrant, and tamoxifen for 48 hours. Five replicates were used for each experiment, and the experiment was repeated three times (**, <i>P</i> ≤ 0.01; ***, <i>P</i> ≤ 0.001; ****, <i>P</i> ≤ 0.0001; ns, nonsignificant). <b>D,</b> Representative image of a clonogenic assay showing the effect of the indicated concentrations of X15695, fulvestrant, and tamoxifen on clonal expansion of tamoxifen-resistant MCF-7 cells. <b>E,</b> Quantification of colonies in the clonogenic assay described in D. <b>F,</b> Representative images of some of the tumors obtained in a mouse MCF-7 tumor xenograft after treatment with vehicle and X15695. <b>G,</b> Tumor volumes of established subcutaneous MCF-7 xenografts treated daily via oral gavage with vehicle or X15695 (30 mg/kg body weight). The values are the mean ± SEM (*, <i>P</i> = 0.05; <i>n</i> = 14). <b>H,</b> Tumor weights at the end of treatment were recorded and presented in the bar chart. The values are the means ± SEM (*, <i>P</i> = 0.05; <i>n</i> = 14). <b>I,</b> Mouse body weights measured twice weekly for all animals over 16 days. Presented are the mouse body weight changes, in percentages, for each group. <b>J,</b> Western blots analysis of ERα and p53 from the extracts of select tumors at the end of treatment. Anti-vinculin antibody was used to determine the level of protein loaded. Quantification of ERα (<b>K</b>) and p53 (<b>L</b>) protein levels in select tumors relative to the vinculin loading control. The results are the mean ± SEM. *, <i>P</i> ≤ 0.05; ***, <i>P</i> ≤ 0.001; <i>n</i> = 12.</p>