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. 2019 Jul 5;10:2988. doi: 10.1038/s41467-019-10926-2

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© The Author(s) 2019

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. 1 — Tkv is induced and internalized in ISCs in response to Ecc15 infection. a Model for the dynamic control of ISC activity by temporal regulation of Sax/Smox signaling and Tkv/Mad signaling in ISCs in response to infection or tissue damage. b Cartoon depicting the gene structure of TkvA-lacZ flies. Transcription from tkv promoter in ISCs (green, arrowheads) under homeostatic conditions (mock) and 4 h after Ecc15 infection. c qPCR analysis of tkv mRNA level in ISCs under mock conditions and after Ecc15 infection (normalized to actin5c). d Cartoon depicting the gene structure of Tkv-3xHA flies. Tkv-3xHA protein was detected within ISCs (esg::Gal4, UAS::GFP, green; Delta+, red) and ISC-derived daughter cells (GFP+, DELTA−), in posterior midgut (PM) of flies at 18 h after Ecc15 infection, determined by immunohistochemistry with rabbit anti-HA antibody. Average HA fluorescence intensity in Delta+ cells was normalized to the mean value of mock. e Time-dependent induction of Tkv in ISCs, measured as the average intensity of Tkv-3xHA in ISCs per PM during the course of a 18 h Ecc15 infection (normalized to the mean value of 0 h). f Median fluorescence of Tkv-3xHA in GFP + ISCs during the course of a 24 h Ecc15 infection, as measured by intracellular Flow Cytometry analysis(normalized to the median value of control samples at 0 h collected on the same day of measurement). g, h Overexpressed Tkv-GFP fusion protein (g) or endogenous Tkv-3xHA protein (h) present as puncta in ISCs (GFP+). Tkv-3xHA puncta, detected by rat anti-HA antibody (used in the rest of our study), corresponded to Rab5+ early endosomes (green, yellow arrowheads) and lysosomes (red, red arrowheads), quantification of which were from single slice images (n = 101 cells from 7 guts) in h. Gain was increased for mock (g) for better visualization. Error bars indicated SEM (b n = 3 flies, d n = 7 flies, e n = 7 flies, f n = 447, 327, 3429, 1713, 1501, 1850, 1884, 2503 cells for 0 h samples; n = 2926, 2450, 1731, 727, 907, 792, 1049 cells for 6 h samples; n = 7230, 2880, 5792, 1209, 1539, 1798, 1636 cells for 12 h samples; n = 2901, 1466, 496, 943, 725, 891 cells for 18 h samples; n = 846, 1319, 1526, 725, 1490 cells for 24 h samples; n = 1148, 4492, 2098, 1954, 2357, 2542, 2666 cells for 3d samples). P values from Student’s t-test (b–d, e) or from one-tailed Wilcoxon rank-sum test (f): ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant. Experiments were repeated 3 times (d, e, g, h)