Endogenous MDM4 is repressed by miR-34a.

<p>(A) MCF7 cells were transfected with expression plasmids for miR-34a or a control mutant of miR-34a. Total RNA was extracted after 48 hours, and RT-qPCR was used to quantify the expression of MDM4 and the known miR-34a target genes CDK6 and CCND1. Expression is relative to the control (miR-34a-mut) transfection condition. Data are the averages of at least three independent experiments. Error bars show 95% confidence intervals. Asterisks and double asterisks indicate t-test values <0.05 and <0.01, respectively, comparing the miR-34a expression plasmid to the control mutant. (B) MCF7 cells were transfected as in (A), and whole cell lysates used for immunoblots to detect MDM4, p53, and beta-actin. Quantification relative to the 34a-mutant condition and normalized to actin are shown below each panel. (C and D) MCF7 cells were transfected with an inhibitor of miR-34a (anti-miR-34a) for 48 hours. RT-qPCR was performed for miR-34a, MDM4, and known miR-34a target genes. (E) Following transfection with miR-34a inhibitor as before, total protein was extracted and immunoblots were performed for the indicated proteins in MCF7.