PTN in the Mammary Gland Figure 5. PTN effect on mammosphere formation. (A) Time course of mammosphere formation day 1, 3 and 5 without and with a blocking anti-PTN antibody 6 recombinant PTN protein. Representative mammospheres that meet the threshold surface area of .900 mm2 are indicated by an arrowhead. Pictures are representative of 3 independent experiments. Scale bar = 50 mm. (B) Quantification of mammospheres (.900 mm2 ) formed per field at day 5. Data are means 6 SE; n = 5 fields/well; 4 wells for each condition. **P,0.01; versus anti-PTN treated cells; ***P,0.001 versus control by ANOVA. (C to F) Immunofluorescence of mammospheres at Day 5 for Ki67 (green)(C), Caspase 3 & DAPI (green & blue) (D), laminin & DAPI (green & blue) or for PTN (grey scale). Staining is shown in mammospheres treated with blocking anti-PTN antibody (anti-PTN) versus control. Scale bar = 20 mm. doi:10.1371/journal.pone.0047876.g005 JNK and p38 MAP kinases. We investigated if PTN inhibits ERK1/2 signaling in MECs. Western blot analysis of whole cell lysates of MECs treated with anti-PTN, the ERK1/2 inhibitor UO126, alone or in combination showed that blocking PTN activity increases phospho-ERK1/2 signaling compared to control and that reverses the inhibitory effect of UO126 to baseline (Figure 7F). We conclude that PTN inhibits ERK1/2 signaling in the mammary epithelial compartment of the mammary gland during ductal outgrowth and branching. PTN activity prevents mammary epithelial cell differentiation As shown above, blocking endogenous PTN activity caused an increase in mammosphere formation and in maturation of the mammary gland during postpuberal development. To further understand the role of PTN in this process, we monitored the impact of anti-PTN treatment on the expression of genes indicative of the progenitor cell status using cultured MECs (CD29, CD49f, SCA-1 and CD10; Figure 8). The respective markers are described in Refs. [59,60,61,62,63,64,65,66] and reviewed further below. There was no significant difference in the mRNA expression levels of the luminal epithelial marker K18 or the mesenchymal marker vimentin when comparing the effect of anti-PTN treated MECs with MECs at passage #1 (Figure 8B). Also, vimentin expression in mammary fibroblasts was ,10-fold higher than in the MECs at passage #1 and we can thus exclude the possibility of a significant fibroblasts contamination of the MEC preparation (Figure 8C). PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47876