U2os human osteosarcoma cells

MDA–MB-231 human breast adenocarcinoma cells, U2OS human osteosarcoma cells, and A431 human epidermoid carcinoma of the skin were obtained from the American Type Culture Collection. MDA–MB-231 and U2Os were cultured in DMEM/10% FBS, and A431 was cultured in DMEM/10% FBS and supplemented with sodium pyruvate. MTLn3 rat mammary adenocarcinoma cells (provided by G. Nicolson, Institute for Molecular Medicine, Huntington Beach, CA; Neri and Nicolson, 1981 (link)) were cultured in DMEM/5% FBS. For EGF stimulation experiments, MDA–MB-231 cells were starved for 16 h in 0.5% FBS/0.8% BSA in DMEM and then for 10 min in L15 media/0.345% BSA. Cells were then stimulated with 2.5 nM EGF (Invitrogen) for 3 min.
For Src inhibition experiments, MDA–MB-231 cells were pretreated with 2 µM PP2 (Sigma-Aldrich) for 18 h, detached, and plated on gelatin-coated plates for additional 4 h in presence of PP2. Cells were then fixed and immunofluorescently labeled for invadopodium precursor markers.

U-2OS human osteosarcoma cells were purchased from the American Type Culture Collection (ATCC) and grown in Iscove's modified Dulbecco's medium (IMDM, EuroClone, Milan, Italy) supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland), 2 mM L-glutamine and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).

All cells were cultured at 37°C and 5% CO₂, and supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. The hTCEpi¹⁴ (link) human corneal epithelial cells, a gift from Dr. James Jester (University of California, Irvine, CA, USA), were cultured in KGM-2 (Lonza, Basel, Switzerland). HCE¹⁵ (link) human corneal epithelial cells, a gift from Dr. Peter Reinach (SUNY College of Optometry, New York, NY, USA), were cultured in Dulbecco's modified Eagle medium (DMEM)/F-12 supplemented with 10% fetal bovine serum (FBS). EPC2¹⁶ (link) human esophageal epithelial cells, a gift from Dr. Anil Rustgi (University of Pennsylvania School of Medicine), were cultured in KSFM (Carlsbad, CA, USA). OKF6¹⁷ (link) human oral epithelial cells, a gift from Dr. James Rheinwald (Harvard Medical School, Cambridge, MA, USA), were cultured in KSFM. E5 cells,¹⁸ (link) which are Vero cells stably expressing HSV-1 ICP4 protein, were a gift from Dr. Neal DeLuca (University of Pittsburgh, Pittsburgh, PA,USA) and were cultured in DMEM supplemented with 10% FBS. HEK293 human embryonic kidney epithelial cells, HeLa human cervical adenocarcinoma cells, U2OS human osteosarcoma cells, H1299 human lung carcinoma cells, and SH-SY5Y human neuroblastoma cells were all obtained from American Type Culture Collection and cultured in DMEM supplemented with 10% FBS.