Cell Lines and Transfections

Human MDA-MB-231 cells and mouse breast cancer cell line 4T1 were purchased commercially from ATCC and periodically verified to be Mycoplasma-negative using the MycoAlert Mycoplasma Detection Kit (Lonza, LT07-218). Cell morphology, growth characteristics, and microarray gene expression analyses were compared with published information to ensure their authenticity. Early passage of cells (<15 passages) was maintained in Dulbecco's modified Eagle medium with 10% fetal bovine serum (FBS) plus 1% penicillin–streptomycin (P/S). Primary tumor cells were cultured in HuMEC-ready medium (Life Technologies) plus 5% FBS and 0.5% P/S in collagen type I (BD Biosciences)–coated plates. ON-TARGETplus siRNAs for ST6GAL1, PODXL, ALCAM1, ECE1, CD97, and ICAM1 and nontargeting control siRNA were purchased from Dharmacon and transfected using Dharmafect (Dharmacon) at 50 nmol/L; for the double knockdown, cells were transfected again after 24 to 48 hours. Transfection efficiency was evaluated by flow cytometry analysis and Western blotting.