Cis diammineplatinum 3 dichloride

Cells were treated for 24 h with each chemotherapeutic agent or solvent control: doxorubicin (MP Biomedicals, LLC, Santa Ana, CA), paclitaxel (Sigma-Aldrich) or cisplatin (cis-Diammineplatinum (III) dichloride, Sigma-Aldrich). Drug concentrations for MMTV-PyMT-derived cells were 2.5 μM paclitaxel, 16 μM cisplatin, or 500 nM doxorubicin. MDA-MB-157 cells were treated with 0.078 μM paclitaxel, 4 μM cisplatin or 12.5 nM doxorubicin. These drug doses were selected after treatment of the MMTV-PyMT;Apc^+/+ cells from 24–72 h showed approximately a 50 % reduction in cell population (data not shown). For the combination treatments, chemical inhibitors were added to the media 18 h after chemotherapeutic agents, resulting in a 6 h treatment with a combination of cisplatin or doxorubicin and 50 μM PP2 (Src inhibitor, Sigma-Aldrich) or 50 μM SP600125 (JNK inhibitor, Sigma-Aldrich). For BrdU incorporation assays, treatment was the same as above with the addition of 5-bromo-2’-deoxyuridine (BrdU, 10 μM, BD Pharmigen, Franklin Lakes, NJ) 8 h after chemotherapeutic agents.

MMTV-PyMT;Apc^+/+ and MMTV-PyMT;Apc^Min/+ cells were isolated from primary tumors from the mouse mammary gland [11 (link)] and grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1:5000 plasmocin (Invivogen, San Diego, CA). All cells were routinely passaged using 0.25% trypsin/EDTA and maintained at 37°C with 5% CO₂. Experiments were performed with cells between passage 10–20. Cells were treated for 24 hours with each chemotherapeutic agent or solvent control: doxorubicin (500 nM MP Biomedicals, LLC, Santa Ana, CA) or cisplatin (16 μM; cis-Diammineplatinum (III) dichloride, Sigma-Aldrich) as described previously [12 (link)]. STAT3 DNA binding was blocked by 24-hour treatment with the small molecule inhibitor A69 [25 (link), 26 (link)].