2.3. Cell lines and culture

A panel of established cell lines originally derived from and representing different grades and stages of human BC were chosen for this study: RT4 (grade-1, non-invasive), RT112 (grade-2, invasive), 5637 (grade-2, invasive), 1376 (grade-3, invasive), T24 (grade-3, invasive), J82 (grade-3, invasive), UMUC3 (high-grade, invasive), TCCsup (grade-4, anaplastic, metastatic), and SCaBER (invasive, squamous cell carcinoma). All cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA), except for RT112 which was obtained from MilliporeSigma (Burlington, MA, USA). The cell lines were free of mycoplasma, proven authentic by the suppliers through the identification of characteristic short-tandem repeats and were used within six months of purchase. The cell lines were cultured in the following media supplemented with 10% fetal bovine serum (FBS): RT4 and T24 in McCoy's 5a medium; RT112 and 5637 in RPMI-1640 Medium; and 1376, TCCsup, J82, UMUC3 and SCaBER in Eagle's Minimum Essential Medium. When tests were performed on the growth factor-mediated effects on PTEN phosphorylation, FBS-free or low-FBS (2%) media were used in comparison with high-FBS (10%) media. All the cell culture reagents were obtained from GIBCO - Thermo Fisher Scientific (Grand Island, NY, USA).