Penicilin streptomycin

Example 8

MCF-7 cells were maintained in high glucose DMEM (Corning cellgro, 15-013-CM) supplemented with 10% FBS, 1× L-Glutamine (29.2 mg/mL), and Penicilin Streptomycin (10,000 I.U./mL Penicillin; 10,000 μg/mL Streptomycin; Corning, 30-009-CI). These cells were plated into 24-well plates at 1×10⁵ cells/well, and the following day Compound 1

[Figure (not displayed)]
was added at 100 nM, alone or in combination with 5 μM of MG-132A (Selleckchem, S2619). The cells were then incubated at 37° C. for 20 hours, and upon cooling, the cells were lysed. The cell lysates were subjected to immunoblotting by standard protocol with primary antibodies of mouse anti-human Estrogen Receptor alpha monoclonal antibody (Santa Cruz Biotechnology Inc., #SC-8002) and 1:1000 goat anti-human actin polyclonal antibody (Santa Cruz Biotechnology Inc. #SC-1616). Western blot results visualized using picoLUCENT™ PLUS-HRP ECL (G Biosciences, 786-165) and LI-COR C digit imaging system. FIG. 9A illustrates the proteasome dependence of Estrogen Receptor alpha degradation induced by compounds of the present disclosure. Similarly, FIG. 9B shows the effect of treatment using Compound 1 alone and in the presence of tamoxifen and expomicin, at 100 nM, 10 μM, and 1 μM respectively.

Example 9

MCF-7 cells were maintained in high glucose DMEM (Corning cellgro, 15-013-CM) supplemented with 10% FBS, 1× L-Glutamine (29.2 mg/mL), and Penicilin Streptomycin (10,000 I.U./mL Penicillin; 10,000 μg/mL Streptomycin; Corning, 30-009-CI). These cells were plated into 24-well plates at 2×10⁴ cells/well, and the following day Compound 1

[Figure (not displayed)]
was added to the medium at various concentrations (1 μM, 100 nM, 10 nM, and 1 nM), with each concentration being added in quadruplicate. At each 24 hour time point, one set of cells at each concentration was trypsinized and mixed with 0.4% trypan blue solution (Corning, 25-900-CI) 1:1 (v/v). The live cells were counted under a microscope using a hemocytometer. The same procedure was repeated using the triple negative breast cancer cell line MDA-MB-231.

FIGS. 11A and 10B demonstrate the compounds of the present disclosure for cells that are selective for Estrogen Receptor alpha positive (10A) over Estrogen Receptor alpha negative (10B).

Example 8

MCF-7 cells were maintained in high glucose DMEM (Corning cellgro, 15-013-CM) supplemented with 10% FBS, 1× L-Glutamine (29.2 mg/mL), and Penicilin Streptomycin (10,000 I.U./mL Penicillin; 10,000 μg/mL Streptomycin; Corning, 30-009-CI). These cells were plated into 24-well plates at 1×10⁵ cells/well, and the following day

[Figure (not displayed)]
was added at 100 nM, alone or in combination with 5 μM of MG-132A (Selleckchem, S2619). The cells were then incubated at 37° C. for 20 hours, and upon cooling, the cells were lysed. The cell lysates were subjected to immunoblotting by standard protocol with primary antibodies of mouse anti-human Estrogen Receptor alpha monoclonal antibody (Santa Cruz Biotechnology Inc., #SC-8002) and 1:1000 goat anti-human actin polyclonal antibody (Santa Cruz Biotechnology Inc. #SC-1616). Western blot results visualized using picoLUCENT™ PLUS-HRP ECL (G Biosciences, 786-165) and LI-COR C digit imaging system. FIG. 9A illustrates the proteasome dependence of Estrogen Receptor alpha degradation induced by compounds of the present disclosure. Similarly, FIG. 9B shows the effect of treatment using Compound 1 alone and in the presence of tamoxifen and expomicin, at 100 nM, 10 μM, and 1 μM respectively.