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. 2022 Oct 21;13(10):885. doi: 10.1038/s41419-022-05308-4

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2022

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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Fig. 5 — a Immunoblotting showing levels of the indicated nuclear and cytoplasmic protein levels in KAL^LU+ and KAL^LU- cells following TNF (10 ng/mL) stimulation. α-Tubulin, a cytosol protein-loading control. b Survival curves of human lung SCC expressing double-high TNFRSF1A and REL levels compared to low TNFRSF1A and REL levels. These data were obtained from the Kaplan–Meier plotter. c Immunoblotting showing UBCH10, E-cadherin, and keratin 5 (K5) levels in KAL^LU+ cells treated with and without TNF (10 ng/mL) treatment for 60 min. β-actin, a protein-loading control. d RT-PCR shows UBCH10 expression in KAL^LU+ cells treated with TNF (10 ng/mL). Mean ± SEM (three repeats per group). **P < 0.01; Student’s t-test. e Immunoblotting showing levels of the indicated protein levels in KAL^LU+ cells following treatment with three different si-UBE2C RNAs (A, B, C, and A + B + C). β-actin, a protein-loading control. f ChIP assay showing the enrichment of p65, c-Rel, and IKKα associated with the Ube2c promoter (p) on regions upstream of −500 bp and −1000 bp in KAL^LU+ cells, immunoprecipitated with p65, c-Rel, and IKKα antibodies. The enrichment was compared to input levels. anti- or Ab, antibody; Neg-Ab, negative-control antibody (Ig); Pos-Ab, positive experimental anti-p65, c-Rel, and IKKα antibody; +IKKα, overexpressed HA-IKKα in KAL^LU+ cells. g ChIP assay showing the enrichment of p65 associated with the Ube2c promoter (p) on regions upstream of −500 bp and −1000 bp, immunoprecipitated with p65 antibody in KAL^LU- and KAL^LU+ cells. The enrichment was compared to input levels. anti- or Ab, antibody; Neg-Ab, negative-control antibody; Pos-Ab, positive p65 antibody; TNF, TNF (10 ng/mL) treatment for 45 min. h ChIP assay showing the enrichment of H3K27ac on the Ube2c promoter (−500 bp) in KAL^LU+ cells treated with and without TNF (10 ng/mL). i ChIP assay showing the enrichment of H3K27ac on the Tnfrsf1a promoter (−500 bp) in KAL^LU- and KAL^LU+ cells. j A working model for how overexpressed TNFR1 promotes and IKKα inhibits Ube2c gene expression, resulting in SCC cell dedifferentiation, aggressive tumors, and metastasis. Silencing TNFR1 or UBCH10 inhibits TNFR1-enhanced tumorigenesis. Arrow, promotion; crossed lines, inhibition.