The labeled cells were washed and then analyzed on a FACS (fluorescence activated cell sorting) Vantage (BD Biosciences). Quantitative real time-polymerase chain reaction (qRT-PCR) After mammosphere cells were sorted, total RNA was extracted by using RNeasy Mini kit (Qiagen, Valencia, CA) and used for qRT-PCR assays in an ABI PRISM 7900HT sequence MK-4827 solubility dmso detection system (ABI, Norwalk, Connecticut). The specific PCR primers were used to detect the presence of Notch2 (F: TATTGATGACTGCCCTAA
CCACA; R: ATAGCCTCCATTGCGGTTGG), β-catenin (F: CCTTTGTCCCGCAA ATCATG; R: ACGTACGGCGCTGGGTATC), CXCR4 (F: TACACCGAGGAAATG GGCTCA; R: TTCTTCACGGAAACAGGGTTC), SDF-1 (F: ATGCCCATGCCGA TTCTTCG; R: GCCGGGCTACAATCTGAAGG) and GAPDH (F: ATGGGGAAGG TGAAGGTCG; R: GGGGTCATTGATGGCAACAATA). GDC-0941 nmr All reactions
were done in a 10-μl reaction volume in triplicate. PCR amplification consisted of 10 min of an initial denaturation step at 95°C, buy BIBW2992 followed by 55 cycles of PCR at 95°C for 30 sec, 56°C for 30 sec and 72°C for 15 sec. Standard curves were generated and the relative amount of target gene mRNA was normalized to GAPDH. Specificity was verified by melt curve analysis and agarose gel electrophoresis. Antagonist reagents Mammosphere cells and monolayer cells of 2 × 105 were cultured in medium (2 ml), and AMD3100, an antagonist of CXCR4, was added to the medium at 1 μg/ml. Then the cells were incubated at 37°C and 5% CO2 for 48 hours. qRT-PCR was used to detect CXCR4 expression in mammosphere cells and monolayer cells. Each experiment was conducted in triplicate. Tissue collection and cell preparation Breast cancer specimens were collected from primary Thymidylate synthase tumors of 4 patients who underwent surgery at Xinhua hospital. Signed informed consent was obtained from all the patients. For comparison, we have also obtained normal tissue from healthy women after plastic surgery. The tissues were minced and dissociated in DMEM/F12 supplemented with 2% bovine serum albumin, 5 mg/ml insulin, 300 U/ml collagenase and 100 U/ml hyaluronidase (all from Sigma)
at 37°C for 18 h. The epithelial-cell-rich pellet was collected by centrifuging at 80 g for 4 min, followed by one wash with DMEM/F12. The supernatant from the first centrifugation was used as a source of mammary stromal fibroblasts. Briefly, the first supernatant were concentrated by centrifugation at 100 g for 10 min, and the obtained mammary stromal fibroblasts were resuspended and cultured in flasks in DMEM/F12 supplemented with 5% fetal bovine serum (Sijiqing, Hangzhou, China) and 5 mg/ml insulin. Differential trypsinization was applied during subculturing to select for the growth of fibroblasts. Immunohistochemistry Coverslips with attached cells were fixed with formaldehyde for 5 min, and then stained with anti-human α-SMA (Dako, Denmark) antibody according to the manufacturer’s instruction.