Figure 1 | Scientific Reports

Figure 1

From: N-Myc-Interacting Protein Negatively Regulates TNF-α-Induced NF-κB Transcriptional Activity by Sequestering NF-κB/p65 in the Cytoplasm

Figure 1

NMI negatively regulates TNF-α-induced IL-6 production. (A) HeLa cells in a 6-well plate were transfected with control or NMI-expression plasmids, and the cells were left untreated or treated with TNF-α (10 ng/ml) for an additional 2 h. The total RNA from these cells was isolated and subjected to qPCR analysis using IL-6, NMI, and GAPDH primers. (B) The IL-6 mRNA expression level was quantified by qPCR analysis. The data represent the level of IL-6 mRNA normalized to the level of GAPDH, which was used as an internal control, and are expressed relative to the level in the control-treated samples that were not stimulated with TNF-α. Results are representative of three independent experiments, and the error bars represent the SD. **p < 0.01. An aliquot of each total cell lysate (TCL) was analyzed by immunoblotting with anti-NMI or anti-actin Abs. (C) HeLa cells were transfected with control or NMI-expression plasmids, and the cells were left untreated or treated with TNF-α (10 ng/ml) for an additional 12 h. The IL-6 levels in the cell culture supernatants were assayed by ELISA. Results are representative of three independent experiments, and the error bars represent the SD. ***p < 0.001. (D) HeLa cells were infected with lentiviruses expressing either control or shRNA targeting NMI. After 48 h, the cells were left untreated or treated with TNF-α (10 ng/ml) for an additional 12 h. The total RNA samples isolated from these cells were subjected to qPCR analysis using IL-6, NMI, or GAPDH primers. (E) Analysis of IL-6 mRNA by quantitative RT-PCR. HeLa-shCtrl and HeLa-shNMI cell lines were left untreated or treated with TNF-α (10 ng/ml) for an additional 12 h, and the mRNA were extracted and subjected to quantitative RT-PCR. The quantified IL-6 transcript levels are shown. The whole-cell extracts were subjected to western blotting using anti-NMI or anti-actin Abs. (F) HeLa-shCtrl and HeLa-shNMI cell lines were left untreated or treated with TNF-α (10 ng/ml) for an additional 12 h. The IL-6 levels in the cell culture supernatants were assayed by ELISA. Results are representative of three independent experiments, and the error bars represent the SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Back to article page