Immortalized human mammary epithelial (HMLE) cells expressing empty vector pWZL (HMLE-vector), Snail (HMLE-Snail) or Twist (HMLE-Twist), and RAS-transformed HMLE (HMLER) cells were maintained as previously described [37 (link), 51 (link)]. HMLE cells were transduced, as previously described [39 (link)], with expression constructs encoding green- or red-fluorescent protein (pMIG or RFP/luciferase, respectively), for use in tube-formation assays. MCF-7, MDA-MB-231, and SUM159 human breast cancer cells were cultured in cell-specific medium as previously detailed [28 (link)]. HMLE-Snail or MDA-MB-231 cells, transduced with shRNA targeting either firefly luciferase (shControl) or FOXC2 (shFOXC2), were previously described [28 (link), 29 (link)].
Cells were treated with vehicle (dH2O) or 100 μM desferrioxamine (DFX) for 24 hours prior to processing for immunofluorescence. For the induction of endothelial differentiation, cells were seeded at a density of 6000 cells/well and cultured for 8–10 days in endothelial cell growth medium (EGM-2 BulletKit™, CC-3162; Lonza Inc, Rockland ME, USA), supplemented with 2% fetal bovine serum and VEGF (designated EGM-2), as previously described [62 (link)].
Cells were treated with vehicle (dH2O) or 100 μM desferrioxamine (DFX) for 24 hours prior to processing for immunofluorescence. For the induction of endothelial differentiation, cells were seeded at a density of 6000 cells/well and cultured for 8–10 days in endothelial cell growth medium (EGM-2 BulletKit™, CC-3162; Lonza Inc, Rockland ME, USA), supplemented with 2% fetal bovine serum and VEGF (designated EGM-2), as previously described [62 (link)].
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