Figure 2

Experimental validation of target gene predictions. Experimental validation of putative target sites on 4128bp (A) and 4169bp (B) fragments of the 10235nt 3^′-UTR of CDK6 (CDK6-1 and CDK6-2, shown as blue bars with indicated putative miRNA binding sites) for miRNAs miR-22, -26a, -26b, -29b, and -137 in HEK293T-Cells. To assess the influence of endogenous miRNAs, empty firefly/renilla dual reporter vector pmirGLO and pmirGLO/CDK6-3^′-UTRs were each transfected into HEK293T-cells. Normalized firefly-activities were compared to those of pairwise co-transfections of these vectors with the miRNA mimic of interest (including an unspecific siRNA negative control) to test for unspecific effects of the given miRNA-mimic on firefly/renilla per se and for validation of the particular target prediction. Dark grey columns show normalized firefly activities from pmirGLO (co)-transfections, light grey columns those from pmirGLO/CDK6-3^′-UTRs (co)-transfections. Percent reductions of firefly activities of pmirGLO/CDK6-3^′-UTRs compared to pmirGLO are given as well as their statistical significancies (at least two biological replicates with four technical replicates each, Student’s t-test, unpaired, ** p ≤ 0.01, ***: p ≤ 0.001). Summarizing results from both CDK6-3^′-UTR fragments, significant regulatory miRNA effects were seen for miR-22, miR-26a, miR-26b, and miR-29b, whereas miR-137 had no significant effect.