Validation and characterization of miR-517a/c. (A) Sequence comparison of the miR-517 family miRNAs. Seed sequences (nucleotides 2 to 8) are highlighted in red. (B and C) HEK293 cells were transfected with the indicated miRNA mimics for 72 hours. mRNA expression of endogenous NF-κB targets IL8 and TNF were determined with quantitative PCR (qPCR) in the absence (B) or presence (C) of TNF. Statistical comparisons are relative to the negative control. (D) HEK293 cells were co-transfected with the indicated miRNA mimics and siRNAs. si-p65-A and si-p65-B are two independent p65 siRNAs. Statistical comparison is relative to cells transfected with miR-517a/c + si-neg. (E) Western blot analysis of nuclear and cytoplasmic protein extracts from HEK293 cells transfected with the indicated miRNAs. Tubulin and Lamin A/C were cytoplasmic and nuclear loading controls, respectively. (F) DNA binding ELISA assay to quantify p65 activation in nuclear extracts from HEK293 cells transfected with the indicated miRNAs. All values are means ± SD. n = 3. *P ≤0.05, **P ≤0.01. miRNAs, micro RNAs; NF-κB, nuclear factor kappa-B; siRNAs, small interfering RNAs; SD, standard deviation; TNF, tumor necrosis factor.