Norcantharidin

The human breast cancer cell lines MCF-7, ZR75-1, T47D and tamoxifen resistant cells MCF-7/TamR were described previously [17 (link)]. Cell lines were authenticated on the basis of viability, recovery, growth, and morphology. The expression status of ER was further confirmed by Western blot before they were used in experiments. All cells were cultured in Dulbecco's Modified Eagle's Media (DMEM) medium containing 10% FBS (Hyclone, Thermo fisher scientific, Florence, KY, USA) at 37°C with 5% CO2 in tissue culture incubators. Norcantharidin (NCTD), 17β-estradiol (E2), propylpyrazoletriol (PPT), diarylpropionitrile (DPN) and 4-hydroxytamoxifen (TAM) were purchased from Sigma (St Louis, MO, USA). The concentrations of these drugs used in this study were according to previous studies [17 (link), 41 (link)].

Cantharidin, Okadaic acid (OA), prostaglandin E2 (PGE2), PD98059, SP600125, RO31-8220, and GF109203X were purchased from Enzo Life Science International (Plymouth Meeting, PA, USA). Norcantharidin (NCTD), Bay11-7082, EF-24, and actinomycin D (ActD) were purchased from Sigma (St. Louis, MO, USA).

MC-LR and MC-RR certified standards, the anti-adda antibody (AD4G2), and the assay kit for detection of MCs/NOD in water by PP2A were purchased from Abraxis, Inc (Warminster, PA, USA). MC-LF, NOD, cantharidin, okadaic acid, and calyculin A standard were purchased from Enzo (Farmingdale, NY, USA). Norcantharidin and phosphate buffered saline, pH 7.4 with Tween 20 (PBS-T) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All MCs and other PP2A inhibitors were stored at −20 °C. The antibody was stored at 4 °C. Acetonitrile was from Pharmaco (Dawsonville, GA, USA). Methanol and formic acid were obtained from Fisher Scientific (Waltham, MA, USA). Life Technologies Corporation (Grand Island, NY, USA) supplied the Dynabeads MyOne Streptavidin T1, Zebaspin 7K molecular weight cut off, 0.5 mL desalting columns, and EZ-link NHS-PEG4-biotin. The Thermomixer C, Protein LoBind 2 mL microcentrifuge tubes, Protein LoBind 1 mL deep well plates, and TwinTec Lo-Bind PCR plates were purchased from Eppendorf (Hauppage, NY, USA). V&P Scientific (San Diego, CA, USA) supplied the 96-well plate magnet (part number VP771HH-MC). The microcentrifuge tube magnet was purchased from Invitrogen (Waltham, MA, USA).

Cells were seeded in 96 well plates and treated with varying concentrations of cantharidin and norcantharidin (Sigma). Stock solutions of cantharidin and norcantharidin were made in DMSO. After 48 hours of treatment, cell viability was analyzed using resazurin as per the manufacturer's recommendations (Fisher Scientific).

Cantharidin, OA and SP600125 were purchased from Enzo Life Sciences International (Plymouth Meeting, PA, USA). NorCantharidin (NCTD) was purchased from Sigma (St. Louis, MO, USA). FH535 was purchased from Millipore (Billerica, MA, USA).

Cantharidin and norCantharidin were obtained from Sigma-Aldrich. All of the drugs were dissolved in DMSO which was used as a solvent control. After 24 h incubation of the cells, the drug treatment was started by removing the old medium (only for 6- and 24-well plates) and adding a fresh medium containing the respective compounds, and all of the drug dilutions contained the same amount of solvent; the 48 h treatments were treated twice every 24 h. Only for the 96-well plates was no medium removed prior to the drug treatment. Afterwards, the plates were incubated for the respective time in a humidified incubator at 37 °C and 5% CO₂.