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. 2021 Jan 7;12(1):42. doi: 10.1038/s41419-020-03299-8

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© The Author(s) 2021

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. 7 — a CHX chase experiments of GPX4 in the control and Bel-7402 cells with TRIB2 knocked out, with or without the simultaneous overexpression of PCBP2. The relative protein levels of GPX4 were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. b TRIB2 and PCBP2 regulated GPX4 ubiquitination. GPX4 or its K48-ubiquitination and total-ubiquitination level in the WCL of Bel-7402 and SMMC-7721 cells with or without TRIB2 knockout in the presence or absence of ectopic expression of PCBP2. The K48-Ub and total-Ub of GPX4 were normalized to that of GPX4 in the GPX4-IPs by ImageJ and indicated below the blots. c PCBP2 acts as the downstream of TRIB2 to prevent the association between GPX4 and proteasome in Bel-7402 and SMMC-7721 cells. The relative GPX4 levels were normalized to PSMB3 in proteasome and normalized to GAPDH at WCL. The relative GPX4 levels were also calculated as the ratio between the one in proteasome and the one at WCL. Besides, the PSMB5 activity was also parallel examined at WCL. Data were analyzed by one-way ANOVA and expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. d, e The TRIB2 DQLVPD element and PCBP2 KH3 domain are critical to maintain GPX4 expression. GPX4 was measured by IB in the Bel-7402 cells transfected with the indicated plasmids. The relative GPX4 levels were normalized to that of GAPDH and indicated below the blots. f GPX4 in control and Bel-7402 cells with or without TRIB2 or PCBP2 knocked out in the presence or absence of overexpressed GPX4. The relative GPX4 levels were normalized to that of GAPDH and indicated below the blots. g Tumor growth in athymic mice inoculated with the same cells as shown in f. The weight and volume of a tumor are presented below the representative tumor image (n = 5/group); Scale bar = 3 mm. Data were analyzed by one-way ANOVA and expressed as mean ± SD. **P < 0.01; ****P < 0.0001. Images of all the immunoblots are representative of three independent experiments.