miR-377 effects on the NF-κB pathway. (A) mel-14PA melanoma cells stably expressing the HTR control vector or a vector expressing miR-377 were co-transfected with NF-κB luciferase vector and renilla vector as an internal control. The resulting cell extracts were analyzed by luciferase reporter assay and values were normalized to the renilla activity. Data is represented as mean ± SEM from three independent experiments. *signifies p < 0.05. (B) Using the Ingenuity Pathways Analysis software suite, we illustrate the network of NFKB1A target genes in melanoma cells over-expressing miR-377. The green and red boxes represent genes that were experimentally found to be down-regulated or up-regulated in mir-377-expressing cells, respectively; the orange and blue lines represent trends of activation or inhibition, respectively, that would be expected following activation of NFKB1A signaling output. The yellow lines represent genes in which a discrepancy exists between the observed change in gene expression and the expected change. (C) The protein levels of Iκβα (NFkB1A) were assessed in miR-377 expressing and control melanoma cell by WB and quantified using densitometry with actin levels serving as internal loading control. (D) The protein levels of Iκβα (NFkB1A) in mel-14PA melanoma cells after treatment with the commercially available inhibitor 5Z-7-oxozeaenol, 4 mM, for 24h. (E) The protein levels of NF-kB in the cytosolic and nuclear cellular fractions were assessed by WB with actin levels (completely absent in the nuclear fraction) serving as internal loading and fractionation control.