Ras and TNF induce phenotype associated with cancer stem cells. A) The effects of Ras or p65 overexpression and TNF treatment on CD44 and CD24 expression status in MCF-10A cells. Activated H-Ras oncogene or the constitutively active p65 subunit of NF-κB was overexpressed by retroviral vector mediated gene transfer. Expression level of Ras in MCF-10A-Ras cells as measured by western blotting is shown (right panel, top) along with number of CD44+/CD24- cells in untreated and TNF treated MCF-10A cells from four experiments (p = 0.03, right bottom panel). MCF-10A-p65 cells have already been described . Cells were treated with 10 ng/ml of TNF for 10 days. B) The effect of Ras overexpression or TNF treatment on the expression of EMT markers in MCF-10A cells. Ras reduced E-cadherin expression, whereas TNF reduced E-cadherin but increased vimentin and αSMA. C) Assay demonstrating the effects of Ras overexpression on mammosphere formation. Cells were plated for 10-days in mammosphere media. Ras increased both the number and the size of mammospheres.