D8418 100ml

Cells were plated in 96‐well plates (Fisher Scientific 07‐200‐95) with 10,000 cells per well and three biological replicates per experiment in phenol‐red free RPMI (Sigma‐Aldrich R8758500ml) with 10% heat‐inactivated charcoal‐stripped FBS (Corning™ 35016CV), 10 nM β‐estradiol (Sigma‐Aldrich E8875‐5G), 0.002% insulin (Sigma‐Aldrich 11376497001), and 50 U/ml penicillin, 50 mg/ml streptomycin (Corning™ MT30001CI), and either DMSO (Sigma‐Aldrich D8418‐100ML), Tamoxifen (Sigma‐Aldrich 579002‐5MG), or Fulvestrant (Sigma‐Aldrich I4409‐25MG). Media was switched out every four days and plates were fixed on days 4 and 8. All plates were stained with crystal violet (Sigma‐Aldrich C0775‐25G) and quantification by spectrophotometric detection at 490 nm using plate reader Molecular Devices Spectramax M3. Ten experimental replicates were performed to obtain parameters (cells per well, estradiol amount, time points) that gave consistent results. Effects were analyzed using GRmetrics version 1.10.0, one‐sided Wilcoxon Rank Sum Test, n = 3.

Flies were treated with EHop-016 (Yong-Can Biotech). The EHop-016 was dissolved in dimethyl sulfoxide (DMSO; Sigma, D8418-100ML) to make a 10-mmol/L stock solution. This solution was stored at −20 °C for later use. Before administration, 4% (m/v) sucrose solution was used to dilute the stock solution to a concentration of 100 µmol/L (working solution). After the flies were starved for 3 h in empty vials, they were fed with the working solution for 5 h. Feeding was carried out once per day until testing.
Mice were treated with EHop-016 (10 mg/kg), CS7170 (50 mg/kg), CS7171 (50 mg/kg), and JKF-034 (40 mg/kg). These drugs were dissolved in saline (0.9% NaCl) containing 0.5% Tween-80 (Sigma P4780). Each mouse received 200 μL of the solution by intragastric administration once per day (administered at 9:00 am). The drug administration was started seven days before the beginning of the Morris water maze (MWM) task and was continued until the end of the assay. All control groups were fed with vehicle solution.