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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. 4 — a RT-PCR showing UBCH10 expression in KAL^LU+ cells treated with si-Control (si-Cont) and si-TNFR1. ***P < 0.001; Student’s t-test (mean ± SD of three samples per group). b Left: Immunoblotting analysis showing levels of the indicated proteins in KAL^LU+ cells treated with si-Control (si-Con) and si-UBCH10 (si-UB). β-actin, a protein-loading control. Right: Immunoblotting analysis showing Twist and UBCH10 levels in KAL^LU+ (-), si-TNFR1 RNA-treated KAL^LU+ cells (si-TNFR1 RNA including si-A, si-B, si-C, and si-ABC), and si-TNFR1 RNA-treated KAL^LU+ cells with reintroduced Ube2c/UBCH10 cDNA. β-actin, a protein-loading control. c Left: Comparison the growth of tumors, derived from KAL^LU+ cells treated with si-Control or si-UBCH10, in nude mice (n = 5/group). Mean ± SD; *P < 0.01; two-way AVOVA test. d Survival curves of lung SCC patients with high TNFRSF1A and UBE2C levels or low TNFRSF1A levels. Lung SCC patient cohorts were obtained from Kaplan–Meier plotter. e Left: Immunoblotting analysis showing levels of K5, E-cadherin, and UBCH10 in KAL^LU+ cells overexpressing a control or HA-IKKα vector. β-actin, a protein-loading control. Right: RT-PCR examines TNF expression in KAL^LU+ cells and KAL^LU+ cells overexpressing HA-IKKα with. Mean ± SEM (three repeats per group). **P < 0.01; Student’s t-test. f Growth curves of KAL^LU+ cells that were transfected with a control vector or HA-IKKα vector (n = 5/group). Mean ± SD; **P < 0.01; two-way AVOVA statistical test. g The sizes of tumors obtained from KAL^LU+ cells treated with the control or HA-IKKα vector (2 × 10⁵ cells per mouse) were subcutaneously injected into nude mice (n = 5/group). Tumor growth was analyzed by a grouped two-way AVOVA statistical test; Mean ± SD; *P < 0.05. h Left: Images showing a liver (top) and lung (bottom) with metastases (Fig. S4a shows H&E histological images). Right: H&E images of liver and lung metastases induced by arrows. Scale bar: 40 μM. i RT-PCR showing UBCH10 mRNA levels in KAL^LU+ cells transfected with IKKα or control vectors (n = 3/group). Mean ± SD; ***P < 0.001; Student’s t-test. j A working model for the TNFR1-UBCH10 pathway predicted to increase c-Myc, Bcl2, Twist1, and Sox2 expression and SCC dedifferentiation. IKKα inhibits UBCH10 expression. Arrow, enhancement; two lines, inhibition. k Human H520 SCC cell line was selected with stem cell markers and used to generate two sub-populations (left). H520 (CD24^loCD44⁺) cells express elevated TNFR1 levels compared to H520 (CD24^hiCD44⁺) cells. Mean ± SD (three repeats per group); ***P < 0.001; Student’s t-test. l H520 (CD24^loCD44⁺) cells generated tumors induced by subcutaneous injections and metastasis to lungs in nude mice (n = 5). Scale bar: 40 μM.