Figure 5

NF-κB inhibits TNF-α expression, and TNF-α increases NF-κB activity. (A) HeLa and C₃₃A cells were transfected with pcDNA3/NF-κB, and total protein from cells was collected to determine the TNF-α levels. The endogenous GAPDH expression levels were used to normalize protein expression. (B) Cells were treated with TNF-α, and Western blot analysis was used to detect NF-κB activity. Cells were collected in PBS, centrifuged, resuspended in 0.4% NP-40, and centrifuged again to obtain nucleoproteins. Nuclear protein CENPA were used to normalize protein expression. (C and D) Cells were seeded on 14-well slides and stimulated with TNF-α (20 ng/mL) for 24 h. Next, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 10% donkey serum. A primary antibody raised against p50 was diluted in 1% normal donkey serum and incubated at 4°C overnight. Cells were washed, and a FITC-labeled donkey anti-rabbit antibody was diluted in 1% normal donkey serum. Finally, DAPI was used to stain the nuclei. All the experiments were performed in triplicate. (E) Verify the role of miR-130a in the NF-kB induction of TNF-α. Cells were transfected with pcDNA3/NF-κB and ASO-miR-130a compare pcDNA3/NF-κB and ASO-NC, the cells were collected totle protein for Western Blot. GAPDH protein expression was used to normalize the endogenous expression data. (F) Cells were treated with TNF-α (20 ng/mL) for 24 h, and RNA was extracted for real-time RT-PCR. U6 snRNA was used to normalize gene expression. (G) The relationship between NF-κB, miR-130a and TNF-α. (*P < 0.05, **P < 0.01).