miR-378 in Differentiation Figure 8. Targeting of O-glycosyltransferase, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNT7) by miR-378. (A) U87 cells transfected with Npnt or Npnt+39 were harvested and subjected to proteomic analysis. Up-regulation of GalNT7 expression was seen in cells transfected with Npnt+39. (B) Computational algorithms were used to predict potential targeting of miR-378 on GalNT7. miR-378 was found to possesses a potential target site on GalNT7 in nucleotides 3360-3378. A fragment of the GalNT7 39UTR harbouring the miR-378 target site was inserted into a luciferase reporter vector. The seed region for miR-378 binding was mutated in GalNT7 as shown (red). (C) Luciferase activity assays were performed by co-transfection of miR-378 with luciferase reporter constructs. Decreased luciferase activity was detected in the construct containing GalNT7 39UTR, which was abolished when the miR-378 target site was mutated. (n = 4, ** p,0.01). (D) Western blot showing decreased expression of GalNT7 in miR-378-transfected cells compared with GFP-transfected cells. (E-F) U87 cells stably transfected with GFP (E) or miR-378 (F) were immunostained with anti-GalNT7 antibody, followed by confocal microscopic visualization. While both types of cells expressed GFP (green), miR-378-transfected cells expressed lower levels of GalNT7 than the GFP-transfected cells (red). As a result, the merged color of miR-378- transfected cells were less yellow. (G) MC3T3 cells were transfected with anti-miR-378 or a control vector at 3 or 6 mg plasmids. Cell lysates were prepared and analyzed on western blot probed with anti-GalNT7 antibody. Transfection with anti-miR-378 enhanced GalNT7 expression. (H) MC3T3 cells were co-transfected with nephronectin and one of the four siRNA oligos against GalNT7 or an oligo at random sequence serving as a negative control. Culture medium was analyzed on western blot probed with the monoclonal antibody 4B6 that recognizes nephronectin. Transfection with siRNAs against GalNT7 decreased the levels of secreted nephronectin. doi:10.1371/journal.pone.0007535.g008 PLoS ONE | www.plosone.org 9 October 2009 | Volume 4 | Issue 10 | e7535