Fig 4.

QKI-5 and QKI-6 abrogate mature miR-7 processing. (A) The levels of miR-7 were measure by real-time qRT-PCR in HEK293 or HEK293 cells stably expressing the indicated plasmids. (B) HEK293 cells stably expressing the indicated hnRNPK minigene were transfected with pcDNA empty vector or expression vectors encoding myc epitope-tagged QKI-5, QKI-6, QKI-7, or QKI-6:V-E (20 μg of plasmid in a 10-cm culture dish). After 48 h, the RNA was extracted and 10 μg of total RNA was loaded and resolved on 12% polyacrylamide TED-urea gels and transferred onto Hybond-N+ membranes. The membranes were hybridized with a U6 antisense and mature miR-7 antisense LNA (Exiqon) probe labeled at the 5′ end with ³²P. The ethidium bromide-stained RNA gel is shown (lanes 1 to 10). The experiment was performed in triplicate, and results were quantified as the means and standard errors of the means. Protein extracts were also prepared and immunoblotted with anti-β-tubulin as a loading control and anti-Myc antibodies to visualize the QKI expression (lanes 11 to 20). The quantification is depicted at the bottom.