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. 2020 Mar 9;11:1270. doi: 10.1038/s41467-020-15003-7

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© The Author(s) 2020

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. 3 — a RNF113A is in both the cytoplasm and the nucleus. A549 cells were treated or not with Cisplatin and WB analyses were carried out with cytoplasmic, nuclear and chromatin-enriched extracts. b, c RNF113A controls the recrutment of NHEJ factors on chromatin upon DNA damage. Control versus RNF113A-depleted A549 cells (b) or control versus RNF113A-overexpressing A549 cells (c) were treated or not with Cisplatin and WB analyses were carried out on chromatin fractions after pre-extraction with the CSK + RNase A buffer. d RNF113A controls the recruitment of pH₂AX on DSBs. ChIP assays were conducted with extracts from control and RNF113A-depleted DIvA U2-OS cells treated or not with 4-hydroxy Tamoxifen (TAM). Primers 183, 906, 307, and 221 are pH₂AX-associated AsiSI sites while primers 811 and 903 are non-associated and serve as negative control³⁶. Immunoprecipitations using anti-IgG antibody served as negative control. The histogram shows recruitment of pH₂AX on indicated sites. Results of two independent experiments (means ± SD, Student t-test, *p < 0.05) are shown. e A N-terminal nuclear localization signal (NLS) controls the nuclear import of RNF113A. The human RNF113A sequence was analyzed using the online NLS prediction algorithm SeqNLS (http://mleg.cse.sc.edu/seqNLS/) and the identified NLS (residues 21–35) is shown in green with a possibility score of 0.882. The subcellular localization of RNF113A constructs was analyzed by immunofluorescence in A549 cells, using DNA-PKcs as a nuclear marker. The percentage of cells showing a cytoplasmic and/or nuclear localization of RNF113A constructs is illustrated in the histogram below. f RNF113A moves in the cytoplasm of apoptotic cells showing some DNA damage. Anti-RNF113A Immunofluorescence analyses were conducted in Cisplatin-treated A549 cells. Cells showing some DNA damage or undergoing cell apoptosis were identifed through anti-pH₂AX and Cyt C stainings, respectively. The histogram show the percentage of cells showing a nuclear or a cytoplasmic and nuclear localization of RNF113A, depending on their status for pH2AX or for Cyt C.