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. 2021 Mar 22;3(1):zcab010. doi: 10.1093/narcan/zcab010

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© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Cancer.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Ubiquitination of TRDMT1 at its K251 is required for cell survival. (A) 3D structure of TRDMT1 using PyMOL marked with glycine 155 (G155) and its adjacent lysine 251 (K251). (B) 293 TRDMT1 KO cells were transfected with GFP-tagged TRDMT1^WT, TRDMT1^G155V, TRDMT1^K251R and TRDMT1^G155V+K251R mutants, respectively. Cells were pulled with anti-GFP and immunoblotted with anti-Ubi. (C) The survival rate of U2OS-TRE TRDMT1 KO cells and U2OS-TRE TRDMT1 KO cells transfected with TRDMT1^WT, TRDMT1^G155V, TRDMT1^K251R after cisplatin treatment at the indicated dose. (D) TRDMT1 KO U2OS cells were transfected with TA-KR and GFP-TRDMT1^WT/GFP-TRDMT1^K251R were exposed to light for 30 min for KR activation and allowed to recover for indicated times (30 min, 4 h, 8 h, 30 h) before fixation. The frequency of cells with GFP-TRDMT1 at TA-KRsites was quantified. (E) Quantification of RAD52 (n = 3, 50 cells per replicate, mean ± SD) and RAD51 at TA-KR sites (n = 20, mean ± SD) after pretreated cells with siTRIM28 or siCtrl. (F) TRDMT1 KO U2OS cells were transfected with TA-KR and FLAG-HA-TRDMT1^WT/ FLAG-HA-TRDMT1^K251R following with treatment with 2D and stained for RAD51. The frequency of RAD51 at TA-KR sites was quantified (n = 3, 50 cells per replicate, mean ± SD). (G) The survival rate of U2OS-TRE TRDMT1 KO cells and U2OS-TRE TRDMT1 KO cells transfected with TRDMT1^WT, TRDMT1^G155V/^K251R after cisplatin treatment at the indicated dose.