Fig. 6: miR-143 induces ERK5 downregulation.

a Enriched genes in miR-143 overexpressing K562-C/EBPα-ER cells after AGO-RIP. Genes detected by RIP-sequencing were plotted against their relative enrichment in AGO2 binding. A 10-fold enrichment in RIP samples was chosen as cut-off for identifying AGO2-bound mRNAs (left). KEGG pathway analysis of genes enriched after AGO-RIP (right). Data represent three independent experiments. b miR-143 and ERK5 mRNA enrichment in AGO-RIP samples. qPCR for miR-143 (upper panel). Bars of the diagram show the fold change of miR-143 expression compared to the input of the control infected cells. Values represent the mean ± SD from three independent experiments. P values are relative to control. *P < 0.05; unpaired two-tailed t-tests. Semiquantitative PCR for ERK5 mRNA and GAPDH as non-binding control in the AGO-RIP samples (lower panels). The gel pictures represent one example of three independent experiments. Numbers below the gel picture indicate the enrichment of ERK5 mRNA in the AGO-RIP of the miR-143 O/E sample compared to the AGO-RIP of the control sample. c Luciferase assay for direct miR-143 binding to the 3´UTR of ERK5. K562-C/EBPα-ER cells were cotransfected using 0.1 µg pRL, 1 µg of either pGL3-ERK5 3´ UTR-WT, pGL3-ERK5 3´UTR-MUT, or pGL3-C/EBPα 3´ UTR in combination with miR-143 or control mimics. Bars represent the luciferase activity for the corresponding vectors. Normalization was done by Renilla luciferase (pRL). Bars in the diagram represent the mean of 3 independent experiments ± SD. *P < 0.05; unpaired two-tailed t-tests. d Western blot for phosphorylated ERK5, ERK5, and c-Myc protein in K562-C/EBPα-ER cells after transient knockdown (left) and overexpression of miR-143 (right) at indicated time points. GAPDH and β-tubulin were used as control. The western blot pictures represent one example of three independent experiments. Numbers below the blots represent the ratio to the respective controls