Figure 6

The Zeb-1 mediated EMT is partially regulated by miRNAs in the miR-23a cluster. (A-C) Phase contrast images showing the morphology of an epithelial MDCK clone (A; 3E11), the same epithelial clone stably over-expressing Zeb-1 (B; 3E11+Zeb1), or a mesenchymal MDCK clone (C; 2F7). (D) Over-expression of Zeb-1 causes the levels of miR-23a, miR-24 and miR-27a to rise dramatically, while over-expression of the miR-23a cluster of genes had no effect on the level of Zeb-1 mRNA, relative to mock-transfected cells. (E) Nedd4L Western: MDCK clone 3E11 has an epithelial morphology and expresses 4 bands that are recognized by an anti-Nedd4l antibody (green). One of these bands (marked with asterisk) was dramatically reduced in MDCK cells with mesenchymal morphology (clone 2F7) or in an epithelial MDCK cell line stably over-expressing Zeb-1 (3E11+Zeb1). The significance of each of the four bands recognized by the anti-Nedd4L antibody remains unclear. However, a similar pattern of bands was observed using a second anti-Nedd4L antibody (data not shown). Anti-Argonaute 2 (Ago2; red) antibody was used as loading control. (F) A hypothetical model for the role of the Zeb-1, the miR-23a cluster, and Nedd4L in TGF-β mediated EMT. The genes and miRNAs involved in the process are listed and the colors indicate the relative expression changes in ILD samples compared to control, red indicates higher level in ILDs, yellow indicates no significant changes, while green represents lower levels in ILD samples compared to control.