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. 2022 Feb 7;13:711. doi: 10.1038/s41467-022-28347-z

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© The Author(s) 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–c Confocal micrographs of germaria from flies maintained for 2 weeks (WK) at 25 °C (a), 18°C (b), or diapause conditions (c). Anti-pMad (green) labels cells with active Dpp signaling from the niche. Anti-γH2AvD (magenta) labels cells with double-strand DNA breaks due to damage [magenta arrowheads in (c)] or meiosis (arrowheads in (b)). d Percentage of H2AvD⁺ cells out of total pMad⁺ cells from 25 °C, 18 °C, and diapause over time. R, recovery. Statistical analysis is by 2-way ANOVA before recovery and 1-way ANOVA with Tukey’s multiple comparisons test after recovery. e, f Ratiometric (405 nm/488 nm) confocal micrographs of germaria from tubulin-mito-roGFP2-Orp1 flies kept for 2 weeks at 25 °C [control (e)] and in diapause (f). g Quantification of ROS in the germarium relative to maximally reduced (with dithiothreitol) or oxidized (with diamide, see methods for details). Statistical analysis is by 1-way ANOVA and Tukey’s multiple comparisons test. ****p < 0.0001. h, i Confocal micrographs of germaria from flies maintained for 2 weeks (WK) at 25 °C (h), or diapause conditions (i). Anti-Vasa labels germline cells (magenta) and 1B1 stains the Hts protein indicating spectrosome/fusome (GSCs are shown by arrowheads). p53 RE GFP-NLS biosensor (green) labels cells with p53 activity (green arrowhead in (i)). j Percentage of p53 RE GFP⁺ GSCs out of total GSCs from 25 °C control and diapause for 2WK. p value from unpaired two-tailed t-test. **p = 0.0095. k Quantification of GSC number/germarium from y, w control flies and p53^−/− in 25 °C control and diapause from 2WK, 6WK, and 6WK recovery (6WK + R). Statistical analysis is by three-way ANOVA before the recovery and one-way ANOVA after recovery. n is the number of GSCs analyzed in (j), in all other cases, n = number of germaria analyzed. Data are mean ± s.e.m. *p = 0.0211; *p = 0.0346; ****p < 0.0001. Scale bars are 20 µm.