Figure 2

Modulation of the ZEB1-miR203-ΔNP63α pathway during luminal epithelial differentiation. The mouse mammary epithelial cell line HC11was plated and on confluence were then withdrawn of EGF for 48 h. Cells were then treated with differentiation inducing media (DIP) for 72 h. Cells were collected for RNA and protein analysis. Total RNA was isolated from cells for qRT-PCR analysis to detect (a) ZEB1 mRNA, (b) mature miR203, (d) HBP1 mRNA and (e) β-casein mRNA. (c) Western blot analysis was performed on HC11 protein lysates to detect ΔNP63α protein expression levels. (f and g) HC11 cells were treated with DIP differentiating media for 72 h in the presence or absence of an adenovirus expressing the ORF of ΔNP63α (lacking the UTR’s), which cause resistance to miR-203 translational inhibition. qRT-PCR analysis were performed on total RNA lysates to detect mRNA expression levels of HBP1 (f), and β-casein (g). Data are means±S.D.; n=3, *P<0.00004, **P<0.02 and ^#P<0.005. (h) Schematic of the regulatory pathway that mediates the downregulation of ΔNP63α protein during luminal mammary cell differentiation