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. 2017 Nov 6;7:14579. doi: 10.1038/s41598-017-15074-5

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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NMI inhibits TNF-α-induced NF-κB activation and knockdown of NMI via shRNA promotes TNF-α-induced NF-κB activation. (A) and (B) NF-κB luciferase reporter assays. HeLa cells (A) or U-2 OS cells (B) in 6-well plates were transiently transfected with a NF-κB luciferase reporter or a NF-κB luciferase reporter together with the FLAG-NMI plasmid. At 24 h posttransfection, the cells were harvested, and the cell lysates were subjected to luciferase assays. The average of the results from three independent experiments is shown. **p < 0.01. (C) and (D) NF-κB luciferase reporter assays. HeLa cells (C) or U-2 OS cells (D) in 6-well plates were transiently transfected with a NF-κB luciferase reporter or a NF-κB luciferase reporter together with the FLAG-NMI plasmid. At 24 h posttransfection, the cells were either untreated or treated with TNF-α for 6 h, and the cell lysates were subjected to luciferase assays. (E) NMI inhibits NF-κB activation induced by RIP, TRADD, or TRAF2. HeLa cells were transfected with the NF-κB luciferase reporter, the RIP/TRADD/TRAF2 plasmids and either control or FLAG-NMI. At 24 h posttransfection, the cell lysates were subjected to luciferase assays. (F) IFNα inhibits NF-κB activity. HeLa cells were transfected with the NF-κB luciferase reporter, and at 24 h posttransfection, the cells were treated as indicated for 6 h (TNF-α: 10 ng/ml, IFNα: 1000 U/ml). The cell lysates were subjected to luciferase assays and western blot analysis. (G) IFNα induces the expression of NMI. HeLa cells were treated with IFNα (1000 U/ml) for the indicated times, and the cell lysates were subjected to western blot analysis with anti-NMI Ab. (H) HeLa-shCtrl or HeLa-shNMI cells were transiently transfected with a NF-κB luciferase reporter. At 24 h posttransfection, the cells were harvested, and the cell lysates were subjected to luciferase assays. (I) HeLa-shCtrl or HeLa-shNMI cells were transfected with a NF-κB luciferase reporter. At 24 h posttransfection, the cells were either untreated or treated with TNF-α for 6 h, and the cell lysates were subjected to luciferase assays. (J) HeLa-shCtrl or HeLa-shNMI cells were transfected with a NF-κB luciferase reporter or the NF-κB luciferase reporter together with the RIP/TRADD/TRAF2 plasmids as indicated, and the cell lysates were then subjected to luciferase assays. All the data shown are the averages of the results from three independent experiments. The data are presented as the mean ± SD of duplicates. *p < 0.05, **p < 0.01, ***p < 0.001.