A2213

Metastatic (MDA-MB 231, 92020424, ECACC, Salisbury, UK) and non-metastatic (MCF-7, ACC115, DSMZ, Braunschweig, Germany) breast cancer cell lines were cultured in RPMI-1640 (F1213, Biochrom AG, Berlin, Germany) medium containing 10 % fetal bovine serum (FBS, S0113, Biochrom), 1 % L-glutamine (K0283, Biochrom) and 1 % Penicillin/Streptomycin (A2213, Biochrom). The cells were maintained at 37 °C in 5 % CO 2 . Sterilized 12 mm diameter circular cover glasses (0.11152 million, Marienfeld Lab., Königshofen, Germany), were placed in 24-well plates. 500 μl FBS was added to each well of the plate for 30 min at room temperature. After aspiration of the FBS from the cells were plated in fl asks and allowed to grow under routine conditions until about 70 % confl uent. After growing the cells on 24 well-plate, the IC 50 dose of Rapamycin (Rapamune, Sirolimus, Pfi zer, Kent, UK) was determined as 1 μg/ml.
The experimental design was determined as the MDA-MB 231-control group (MDA-MB 231 metastatic breast cancer cells without exposure to any drug), MCF-7-control group (MCF-7 non-metastatic breast cancer cells without exposure to any drug); MDA-MB 231-Rapamycin group (MDA-MB 231 metastatic breast cancer cells treated with 1 ug/ml Rapamycin) and MCF-7-Rapamycin group (MCF-7 non-metastatic breast cancer cells treated with 1 ug/ml Rapamycin) evaluated the effect of 24-hour Rapamycin treatment.

Primary Dermal Fibroblasts Mouse dermal fibroblasts were isolated, as previously described, and used in experiments up to passage 6. 50, 67 Briefly, the entire trunk skin of postnatal day 3 C57BL/6N mice and dKO mice was removed and incubated overnight at 37 C with 0.05% trypsin (15090-046; Life Technologies)-EDTA (0.02%) in PBS. On manual detachment from the epidermis, the dermis was minced and cells were liberated by incubation with Dulbecco's modified essential medium (41965; Life Technologies) containing collagenase I (400 U/mL; LS004196; Worthington, Lakewood, NJ) for 1 hour at 37 C. Debris was removed by straining through 70-mm filters (352350; BD Falcon, Heidelberg, Germany), and the cells isolated from one mouse were plated on a 10-cm dish in Dulbecco's modified essential medium supplemented with 10% fetal calf serum (10270; Life Technologies), 2 mmol/L L-glutamine (K0283; Biochrom, Berlin, Germany), and 10,000 U/mL penicillin and 10,000 mg/mL streptomycin (A2213; Biochrom).