PTN in the Mammary Gland Figure 4. PTN protein expression and function in mammary epithelial cells (MECs). (A) Immunofluorescence (IF) staining of MECs grown on poly-D-lysine coated slides. PTN (green), DAPI (blue), Phalloidin (red). Images are shown as merged images of anti-PTN with DAPI and anti-PTN with Phalloidin staining. Scale Bar = 20 mm. (B, C) Real-time impedance sensing of MECs growth relative to stimulation time (20 hours) with anti-PTN blocking antibody (anti-PTN), recombinant PTN (PTN), or antibody elution buffer (Control) (B), bFGF or resuspension buffer (C). (D) Real-time impedance sensing of MECs migration relative to 30 hours from plating and four hours from scratching. MECs were treated with anti-PTN blocking antibody (anti-PTN), recombinant PTN (PTN), alone or in combination or antibody elution buffer (Control). (E) Real-time impedance sensing of MECs invasion relative to 30 minutes from plating. MECs were treated with anti-PTN blocking antibody (anti-PTN), or antibody elution buffer (Control) (E), recombinant PTN (PTN) or PTN resuspension buffer (Control) (F). The data are representative of at least three independent experiments done in duplicates. ***P,0.001 versus control by ANOVA. doi:10.1371/journal.pone.0047876.g004 was accompanied by a change in pathway activation. The analysis of whole mammary gland lysates of anti-PTN treated versus control mice showed an increase in phospho-ERK1/2 but not in the phospho-p38 and phospho-JNK pathway (Figure 7). This suggests that PTN inhibits ERK1/2 signaling in the mammary gland during ductal outgrowth and branching but does not impact PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47876