Comparison of WIT medium containing EGF or NRG1/AREG. Human mammary epithelial cells were infected with vectors expressing TERT, BMI1, CCND1 and MYC. A. Western blots showing that the transgenes are expressed. The cells in medium containing AREG and NRG1 (svWIT) have luminal characteristics. The cells in medium containing EGF (pWIT) have stronger phosphorylation of EGFR. B. Photomicrographs showing that cells in pWIT are a mixture of spindle shaped and rounder cells, whereas cells in svWIT have a more uniform cobblestone appearance. Scale bars 250 μm. C. Flow cytometry shows that cells in svWIT have a putative luminal progenitor phenotype (CD49+/EPCAM+) whereas cells in pWIT are a mixture of putative luminal progenitors and myoepithelial cells. The quadrants were defined by the isotype controls.