Repression of Tumors in miR-143 Transgenic Mice Figure 5. Analysis in human colon cancer cells. A-C) Western blot analysis of the transfectants. Whole cell extracts were immunoblotted with the indicated antibodies. A) DLD-1 cells in 24-well culture plates were transfected with 1 mg of dominant negative MEK5 (HA-MEK5A) or 1 mg of constitutive active MEK5 (HA-MEK5D). pcDNA3 vector was transfected as a mock. B, C) DLD-1 cells in 24-well culture plates were transfected with 20 p mol of negative control siRNA (Mock), each miRNA mimic or siRNAs for ERK5 and c-Myc. 10 p mol of each miRNA mimic was transfected for a combination of miR-143 and miR-145. sic-Myc #1,3 represent siRNAs for c-Myc. Lower and upper arrows indicate p72 and its alternatively spliced form p82, respectively. D) Schematic depiction of pGL3-Promoter plasmids fused to the 39UTR fragment of p72 containing a potential target site of miR-145 and its mutant. Mutated sequences are boxed. Complementary pairs of miR-145 and its target within the seed sequence are shown as vertical bars. E) % inhibition of luciferase reporter activity of the wild and mutant transfectants by miR-145 mimic is shown. F) qRT-PCR of miR-145 (F), miR-143 (G) and pri-miR-145 (H) in DLD-1 cells and Lovo cells. Cells in 24-well culture plates were transfected with 20 p mol of negative control siRNA (open bar), or siRNAs for c-Myc#1 (gray bar). The data are representative of at least two independent experiments and are presented as the mean 6 SEM. doi:10.1371/journal.pone.0042137.g005 miR-143 and miR-145 in colon cancer cells might be due to insufficient c-Myc inhibition. Therefore, it is likely that the suppression of c-Myc at least in part contributes to the downregulation of p68 expression in the small intestine tumors of the transgenic mice. These findings suggest that miR-143 and miR-145 might act in concert to inhibit both ERK5/c-Myc and p68/p72/b-catenin signaling, and thereby suppress the expres- sion of cyclin D1, c-jun and c-Myc itself. PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42137