Figure 3: NF-κB p65 binds miR-503 promoter and regulates its transcription.

(a) HUVECs were treated with HG (control: L-Glucose) or transduced with Ad.p75 (control: Ad.Null). Representative western blot images and protein quantification of NF-κB p65, Tubulin and Laminin in nuclear and cytoplasmatic cell extracts *P<0.05, **P<0.01 versus Cont (n=3). (b) Sonicated chromatin of samples prepared as above was subjected to ChIP using normal mouse IgG or anti-NF-κB p65 antibody. Nuclear translocation of NF-κB p65 was verified with qPCR using primers for the IKBα promoter as a positive locus. (c) NF-κB p65 enrichment of miR-503 promoter (−3,480 bp from TSS). (d) Occupancy of H3K4me3 in the region of the NF-κB p65-binding sequence within the miR-503 promoter. For b–d, IgG-precipitated samples were used as negative control. Data are presented as % input for each IP sample relative to the input chromatin (1%) for each amplicon and ChIP sample as indicated; *P<0.05 versus Cont or Ad.Null; **P<0.01 versus Cont (n=3). (e) HUVECs were transfected with various luciferase reporter constructs spanning the putative NF-κB-binding sites of the miR-503 promoter and treated in the above conditions. Luciferase activity was measured and presented as a fold-change compared with osmotic control or Ad.Null. *P<0.05 versus empty vector (n=5). Unpaired two-tailed Student’s t-test or Mann–Whitney nonparametric test was applied. All values are mean±s.e.m. of three independent experiments.