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. 2020 Jan 9;10:103. doi: 10.1038/s41598-019-57096-1

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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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Localization and expression of FLAG::FCD-2 after ICL damage in the C. elegans germline. (a) Representative image of gonad dissected from FLAG::fcd-2 transgenic hermaphrodite worm and co-stained with anti-FLAG antibody (green), anti-RAD-51 (in red) and DAPI (blue). Bar, 10 μm. (b) Representative image of gonad dissected from FLAG::fcd-2 transgenic hermaphrodite worm and co-stained with anti-FLAG antibody (green), anti-RAD-51 (in red) and DAPI (blue) after 48 hours CDDP treatment. Bar, 10 μm. Regions 1 and 2 consist of mitotic nuclei, region 3 consists of meiotic nuclei in transition zone (leptotene and zygotene). The regions 4, 5 and 6 correspond to early, middle and late pachytene respectively. The region 7 consists of diplotene nuclei. (c) Strip chart of FCD-2 foci in mitotic zone and early pachytene of FLAG::fcd-2 strain before and after 48 hours CDDP treatment. Each dot indicates the number of FCD-2 foci per nucleus. Red bar indicates the average for each different condition. Error bars correspond to SD calculated from at least three independent experiments. P value (Student’s t-test): ***P < 0.0001. (d) Representative images of diplotene nuclei in FLAG::fcd-2 transgenic hermaphrodites co-stained with anti-FLAG antibody (green), and DAPI (blue), untreated or treated with CDDP. Bar, 10 μm. (e) Western blot analysis of increasing amounts of protein extracts prepared from synchronized worms (FLAG::fcd-2 strain) treated or not with CDDP. The same filter was probed with antibodies against the indicated proteins. The panel shows different parts of the same filter probed with indicated antibodies. Anti -Actin was used as loading control. Data presented are representative of at least three independent experiments performed with at least three different amount of Protein extract. (f) Quantitation of FCD-2 levels normalized against Actin. Signal intensity of each band was measured using Quantity One software (Biorad). The y-axis represents the relative expression of FCD-2 in protein extracts from untreated worms and after CDDP treatment. Bars represent the means ± error standard from at least three independent experiments performed with three different amount of Protein extract for each experiment. P value (Student’s t-test): n.s. = no significant difference.