Doxorubicin and TGFb Inhibitor Figure 3. Inhibition of Doxorubicin- and TGFb-induced EMT by TbRI-KI in murine 4T1 cells. A. Representative pictures of 4T1 cells with mesenchymal morphology after treatment with TGFb1 (2 ng/ml) or doxorubicin (25 nM) for 7 days (6100 magnification). B. 4T1 cells were treated with or without TGFb1 (2 ng/ml) or TbRI-KI (500 nM) for 7 days. Cell lysates were analyzed by western blotting with antibody to Vimentin and tubulin. C. 4T1 cells were treated with TGFb1 (2 ng/ml) or doxorubicin (25 nM) in the presence or absence of TbRI-KI (500 nM) for 7 days. The treated and non-treated 4T1 cells were harvested and about 46103 cells were seeded on a 8-well Lab-Tek chambered coverglass. After 24 hours, the cells were stained with Rhodamine phalloidin for F-actin or an antibody to Snail. Fluorescent images were taken with a confocal microscope. doi:10.1371/journal.pone.0010365.g003 spheres’. Each of these spheroid represents a mammary stem/ early progenitor cell which undergoes limited self-renewal and then gives rise to mammary progenitors capable of multi-lineage differentiation. It has been shown that mammosphere cultures from tumor cell lines and human breast metastasis specimens enrich for tumor initiating cells [40]. Induction of EMT has been shown to promote mammosphere formation in primary human mammary epithelial cells after prolonged treatment with TGFb [26]. We also observed that treatment of 4T1 cells with TGFb1 (0.05 and 0.1 ng/ml) for 12 days stimulated the formation of mammospheres (Figure S4). We have used this model to study whether doxorubicin induced EMT can promote the formation of mammospheres which represents self-renewing stem/early pro- genitor cells. We found a 5-fold increase in the number of mammospheres formed by the treatment of 4T1 cells with 25 nM doxorubicin for 7 days, in comparison to the mammospheres formed by the non-treated cells. This enhanced formation of mammospheres by doxorubicin was drastically reduced by the addition of TbR1-KI (Figure 5A and Figure 5B). Thus, our in vitro results revealed that the chemotherapeutic agent doxorubicin can induce TGFb signaling, promote EMT and generate cells with drug resistant stem cell phenotypes in murine PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10365