Toremifene

Stock solutions of drugs were prepared in dimethyl sulfoxide (DMSO) and stored as frozen aliquots until use. Cells were incubated with 3.47 (Microbiotix), amiodarone (Sigma), bepridil (Sigma), clomifene (Sigma), sertraline (Toronto Research Chemicals), or toremifene (Sigma) for 1 h or 16 h at 37°C with concentrations as indicated. Incubation of cells with E-64d (Peptides International) was extended to 6 h at 37°C, while cells were not preincubated with U18666A (Calbiochem) for PLA, but preincubated for 1 h or 16 h at 37°C for additional described experiments at concentrations indicated. Inhibitors were maintained at the same concentrations during virus spinoculation and virus entry into cells.

All SERMs were obtained from the following commercial sources. tamoxifen and Y-134 were purchased from Tocris Bioscience (Bristol, United Kingdom). N-desmethyl tamoxifen, 4-hydroxy tamoxifen, endoxifen, SO₄-tamoxifen, toremifene, 4-hydroxy toremifene, OSP, RAL, lasofoxifene, NAF, and BAZ were all obtained from Sigma Aldrich (St. Louis, MO, USA). Acolbifene was procured from AdooQ Bioscience (Irvine, CA, USA).
AM-630, AM-251, DAMGO, and WIN-55,212-2 were purchased from Tocris Bioscience. CP-55,940 was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). [³⁵S]GTPγS (1250 Ci/mmol) was procured from American Radiolabeled Chemicals (St. Louis, MO, USA) and [³H]CP-55,940 (131.4 Ci/mmol) was purchased from PerkinElmer (Waltham, MA, USA).
All other reagents were purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA). All compounds were dissolved in 100% DMSO to produce a stock concentration of 10 mM.

The following drugs were used to perform the assays: Tamoxifen (10 µM, SIGMA 3–48 h), Toremifene (10 µM, SIGMA 48 h) and Ospemifene (10 µM, SIGMA 48 h).
Screening and dose‐response: Cells were plated on 384‐well plates (2 × 10⁴ cells per well). After 24 h, cells were treated with 10 µM compounds or 0.1% dimethyl sulfoxide (DMSO) in complete medium. The Prestwick Library consists of 1,280 FDA‐approved drugs, all off‐patent, dissolved in DMSO. The drugs from the 96‐well source plate were diluted and compacted in 384‐well plates to a concentration of 100 μM in the DMEM medium (working plate). To study the effect of the drugs, 5 μl of the drugs at 100 μM in DMEM medium were added to plates containing 45 μl of medium (10 μM final drug concentration with 0.1% DMSO). As a positive control of Gb3 reduction, we used the glucosylceramide synthase inhibitor PDMP.
For the dose–response confirmation test, compounds were serially diluted from 10 mM stock into complete medium and added to plates starting at 30 to 0.1 µM. The final concentration of DMSO did not exceed 0.3% in the dose–response assays.
Cells were incubated together with drugs 48 h at 37°C and 5% CO₂.

Breast cancer cell lines MCF7 and Bcap37 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Both cell lines were ER positive. Cells were grown in normal DMEM with 10% FBS, 100 unit/mL penicillin and 100 µg/mL streptomycin. Prior to all experiments, cells were pretreated for at least 1 week with an estrogen-free medium (phenol red-free DMEM with 10% dextran-coated charcoal serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 0.5 mM sodium pyruvate, and 2 mM l-glutamine) to prevent the effects of serum-derived estrogenic compounds. Cell transfection was carried out using Lipofectamine 2000 (Cat. 11668, Invitrogen) according to the manufacturer's instructions.
E2 (E8875, Sigma) was prepared as a 10 mM solution with ethanol and stored at −80°C. Tamoxifen (T5648, Sigma) or toremifene (T7204, Sigma) was dissolved in 10 mM DMSO. Phps1 (P0039, Sigma,) was dissolved in PBS solution. NSC87877 (565851, Millipore) and phps4 (a gift from Dr. Yuehai Ke (Department of Medicine, Zhejiang University) were dissolved in DMSO. Plasmids expressing siRNAs of Shp2 Plvth-H1, Plvth-H2, and Plvth-H3, as well as a control plasmid Plvth were also prepared as previously described [53] (link). Unless otherwise indicated, all other chemicals were obtained from Sigma/Fisher.