PTN in the Mammary Gland Figure 2. Mammary gland tissue distribution of PTN mRNA. (A) In situ hybridization (ISH) with PTN mRNA antisense and sense probes. Red squares indicate the areas shown at higher magnification. Scale Bars = 25 mm and 100 mm respectively. Red arrowheads indicate PTN mRNA expression in mammary epithelial cells. (B) Northern blot for PTN mRNA in virgin mammary glands fractionated into epithelial and stromal cells. doi:10.1371/journal.pone.0047876.g002 that PTN activity regulates MECs migration and invasion (Figure 4D,E,F). Treatment with the anti-PTN antibody inhibited MECs migration (Figure 4D; p,0.001). After an initial inhibitory effect on MECs migration (p,0.001), the addition of recombinant PTN restored MECs migration to control levels after 12 hours and reversed the effect of the blocking antibody on MECs migration. These findings suggest that MECs motility is dependent on endogenous PTN activity. Invasion studies measured by real-time monitoring electric impedance show that PTN activity maintains MECs invasion. Overall these findings suggest a role for PTN as a promoter of MECs motility and invasion. Growing MECs in a three-dimensional mesh such as Matrigel has been shown to maintain the signaling cues for cellular organization and differentiation that are more representative of in vivo conditions than when grown on a plastic surface [49,50,51,52]. MECs treated with the blocking antibody formed a significantly higher number of mammospheres per field (Figure 5A,B; p,0.001) compared to controls. Adding recombinant PTN protein to the MECs treated with the anti-PTN antibody reversed the effect of the antibody (Figure 5B; p,0.01). Also, immunofluorescence showed PTN staining in the control and not in the anti-PTN treated mammo- spheres as evidence of sufficient dosing of the anti-PTN antibody (Figure 5F). This confirms that mammosphere formation potential of MECs is controlled by endogenous PTN activity. Immunoflu- orescence staining for the proliferation marker Ki67 shows not apparent difference between control and anti-PTN treated spheres (Figure 5C). This concords with what we observed in the proliferation studies with MECs grown on plastic (Figure 4B). Immunofluorescence staining for the apoptosis marker Caspase 3 suggests that PTN does not regulate MECs apoptosis (Figure 5D). We observed that MECs were able to form mammospheres with a lumen surrounded by a monolayer of polarized cells and laminin PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47876