The anti-NK1 peptide was against the carboxyterminal tail of the NK-1 receptor, which corresponds to amino acids 393-407 of the NK1-FL receptor. Reagent A (Polymer Selleckchem KPT330 enhancer), Reagent B (polymerized horseradish peroxidase-anti mouse/rabbit lgG), citrate buffer (pH = 6.0), normal non-immune goat serum (10%), and DAB were purchased from Maixin (Fuzhou, China). SMSP was obtained from Tocris (Avonmouth, UK). SR140333 was kindly provided by Sanofi-Aventis-Chilly-Mazarin. FBS, DMEM (high glucose), trypsin-EDTA (0.05% trypsin 0.53 mM EDTA) were purchased from Gibco (California, USA). MTT, DMSO and Hoechst33258 were purchased from Sigma (Saint Louis, USA). 25 cm2 culture flakes, 96-well

culture plates and 15 mL centrifuge tubes were purchased from Corning (New York, USA). All breast tissues were obtained from Qingdao Municipal Hospital. The patients

providing the tissues did not receive prior treatment with anticancer agents. The study was approved by the institutional review board of Qingdao University. The following tumors were investigated: infiltrating ductal carcinoma (n = 89), infiltrating lobular carcinoma (n = 14). The human breast cancer cell line T47D was purchased from Chinese Type Culture Collection (Shanghai, China). The T47D cells were seeded in 25 cm2 culture flakes and maintained with DMEM supplemented with 10% FBS. The medium was renewed every two days and the cells were passaged by treatment with trypsin-EDTA on the six day after seeding. On the third day T47D JAK inhibitor cells entered exponential phase. Cells were incubated at 37°C in CO2 incubator (SHEL LAB, Oregon, USA) containing 5% CO2. All T47D cells were dissociated by treatment with trypsin-EDTA at 80-90% cell confluence and inoculated at a density of 105cells/mL in C-X-C chemokine receptor type 7 (CXCR-7) 6-well plates which contained cover slips. The medium was renewed after two days and the cover slips were extracted on the fourth day, then the specimens were put into acetone (4°C) to

fix for 15 minutes. Immunohistochemistry All tissue specimens were fixed in formalin and embedded in paraffin. Seven-μm paraffin RSL3 in vitro sections were cut and floated onto polylysine adhered slides. The sections were dewaxed in xylene and rinsed in alcohol and graded alcohol/water mixtures. The immunohistochemical staning was performed using Elivision™ plus two-step System. Briefly, all sections were incubated with 3% hydrogen peroxide for 15 minutes to block endogenous peroxidase activity at first. The sections were subsequently treated in a microwave oven twice for 6 minutes in citrate buffer at 600W to undergo antigen repairing. After blocking with goat serum for 30 minutes, rabbit anti-human NK-1 was applied on the sections at the dilution of 1: 700 for 90 minutes at room temperature. After rinsing, staining was performed with Reagent A and Reagent B subsequently. The color was developed by reacting with DAB. Sections were then counterstained with hematoxylin, dehydrated, cleared and coverslipped.