Supplemental Experimental Procedures

The HMLE-Snail-ER and HMLE-Twist-ER cells were generated by infecting the HMLE cells with pWZL-Snail-ER or pWZL-Twist-ER vectors followed by selection with 5 ng/ml of blasticidin. HMLE-Snail-Ras, HMLE-Twist-Ras, and HMLE-Vector-Ras cells were generated by infecting immortalized human mammary epithelial cells (Elenbaas et al., 2001) with retroviral vectors expressing Snail or Twist or the control vector and selecting with 2µg/ml puromycin. These cells were then transformed by introduction of a pWZL retroviral vector expressing the V12H-RAS oncogene followed by selection with 4µg/ml of blasticidin. Mammosphere culture Mammosphere culture was performed as described in Dontu et al 2003 (Dontu et al., 2003), except that the culture medium contained 1 % methyl cellulose to prevent cell aggregation. The mammospheres were cultured for 7-10 days. Then the mammospheres with diameter>75 µm were counted. For serial passages, mammosphere were harvested using 70 µm cell strainers after culturing mammospheres for 10 days. The mammospheres were dissociated to single cells with trypsin, and 500 dissociated cells were plated in a 96-well plate and cultured for 10 days. The mammospheres were then counted. The individual mammospheres were found to be derived from single cells, because mixing of equal numbers of DiI (Red)- or DiO (Green)-labelled cells prior to performing the mammospheres containing only one or the other label