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. 2021 Sep 1;12:5217. doi: 10.1038/s41467-021-25282-3

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© The Author(s) 2021

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. 2 — a Reporter strategies. Green horizontal bars, duration of CTL contacts. Panel 1: CTL-mediated perforin pores visualized as Ca²⁺ influx into B16F10/OVA target cells using the GCaMP6s reporter. Time-resolved intensity plot of GCaMP6s event in the target cell cytosol followed by survival. Green, OT1 CTL (GFP); Fire LUT, Ca²⁺ level (GCaMP6s). Panel 2: CTL-mediated structural damage of the nuclear lamina monitored as NLS-GFP leakage into the cytosol. Nuclear and cytosolic GFP intensity plotted over time. Green, NLS-GFP; red: Histone-2B-mCherry; arrowhead: monitored cell. Asterisks, nuclear leakage events. Panel 3: CTL-mediated DNA damage response plotted as 53BP1trunc-Apple focalization in the nucleus over time. Fire LUT, 53BP1trunc-Apple. Insets, zooms of reversibility of 53BP1 foci. Scale bars, 10 µm. Image sequences in panels 1 and 2 show representative examples from experimental datasets shown in b–d. b Percentage of contacts of wt and perforin-deficient CTL with B16F10/OVA cells inducing Ca²⁺ events, NLS-GFP cytosolic leakage, and 53BP1trunc-Apple foci in target cells. Data show the mean ± SD from N = 3 (GCaMP6s/wt, NLS-GFP/wt/prf1−/−), 5 (53BP1trunc-Apple/wt), and triplicate movies from 1 (GCaMP6s/prf1−/−, NLS-GFP/prf1−/−) independent experiments. p-Values, two-tailed Mann–Whitney test comparing wt and prf1−/− datasets. c Recovery times from initiation to termination of GCaMP6s, NLS-GFP, and 53BP1trunc-Apple reporter activity. Data show the medians with whiskers from minimum to maximum values derived from N = 32 (53BP1trunc-Apple), 40 (NLS-GFP), and 71 (GCaMP6s) individual events pooled from 3 (NLS-GFP, GCaMP6s) and 5 (53BP1trunc-Apple) independent experiments. p-Value, Kruskal–Wallis test comparing all groups corrected by Dunn’s multiple comparisons test. d Percentage of cells with sublethal damage event, which fully resolved. Data show the mean ± SD (five independent experiments per reporter) for an 1:2 ET ratio. Source data are provided as a Source Data file.