Cyclophosphamide

D2F2/E2 cells, a mouse mammary tumor line that has been stably transfected with a vector expressing the human HER2/neu gene and its parent cell line, D2F2 were a generous gift from Dr. Wei-Zen Wei, (Karmanos Cancer Institute, Wayne State University, Detroit, MI USA). Early passage cells were frozen and periodically thawed for experimental use or restocking. Mycoplasma testing was negative using the Impact III PCR profile from IDEXX (RADIL, Columbia, MO, USA). Anti-CTLA4 monoclonal antibody (mAb 9H10) was obtained commercially (BioXcell Fermentation/Purification Services #BE0131, West Lebanon, NH, USA) as was cyclophosphamide (Bristol-Myers Squibb Co., Princeton, NJ, USA). Mice were 8 to 20 weeks of age and weighed 20-25 g. Thy 1.2 BALB/c were obtained from Taconic (Hudson, NY). Animal studies were approved by the University of Pittsburgh institutional Animal Care and Use Committee (IACUC Protocol #: 21028761).

Mice were injected subcutaneously with 100 μl of corn oil containing 2 μg estradiol (Sigma, St. Louis, MO) every day for 7 days unless otherwise noted. Oil treated mice and E2 treated mice were caged separately. Poly I:C (Amersham, Piscataway, NJ) was resuspended in PBS at 50 μg/ml, and mice were injected intraperitoneally with 0.5 μg/gram of body mass every other day for 6 days. To measure protein translation, mice were injected intraperitoneally with 500 μg of puromycin (ThermoFisher Scientific, Waltham, MA) and sacrified one hour later. To promote HSC activation and mobilization, mice were intraperitoneally injected with 4 mg of cyclophosphamide (Bristol Myers Squibb, NY) on day −1, and then injected subcutaneously on successive days with 5 μg of human G-CSF (Amgen, Thousand Oaks, CA). Mice were sacrificed on day 3 or 6.