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. 2023 Feb 8;614(7949):767–773. doi: 10.1038/s41586-023-05710-8

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 10 — a, Line chart showing growth curve analysis of IMR90^E6E7 expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag lacking Zα2 with either TRF1(ΔN) or VP16-TRF1(ΔN). Fresh media with doxycycline (1 μg ml⁻¹) was added every 2 days. Two independent experiments were performed. b, Scatter plots showing RT-qPCR analysis of ISG levels in cells as in a. Expression levels were normalized to control cells expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag lacking Zα2 with TRF1(ΔN). RNA extracts were collected at day 20 of the experiment. Bars represent mean ± s.d. from technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, *** p < 0.001. Two independent experiments were performed. c, Line chart showing growth curve analysis of IMR90^E6E7 expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag containing point mutations in Zα2, with either TRF1(ΔN) or VP16-TRF1(ΔN). Fresh media including 1 μg ml⁻¹ of doxycycline was added every 2 days. Two independent experiments were performed. d, Scatter plots showing RT-qPCR analysis of ISG levels in cells as in c. Expression levels were normalized to control cells expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag containing point mutations in Zα2 with TRF1(ΔN). RNA extracts were collected at day 30 of the experiment. Bars represent mean ± s.d. from technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, *** p < 0.001. Two independent experiments were performed. e, Experimental timeline. Growing (PD35) IMR90^E6E7 expressing WT ZBP1(S)-Flag were treated with doxycycline (1 μg ml⁻¹) prior to four sequential siRNA transfections. Two individual siRNAs targeting TERRA were used. Samples were collected at days 2,4,6, and 8 post-doxycycline. f, RNA-dot blot performed on RNA isolated from total cell lysates at day 8 post-doxycycline as shown in the timeline of the experiment, using ³²P-dCTP-labelled probes targeting either TERRA or GAPDH transcripts. RNase A treatment was used to assess possible DNA contamination. GAPDH loading control. Three independent experiments were performed. g, Western blotting performed on protein extracts collected at days 2,4,6, and 8 post-doxycycline treatment as shown in e. GAPDH loading control. Two independent experiments were performed. Abbreviations: ZBP1(S): short isoform; WT: wild-type; CTR: control; PDs: population doublings.

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