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. 2021 Jun 16;12:3655. doi: 10.1038/s41467-021-23969-1

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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. 5 — a RNA uptake in B220⁺CD11c⁺NK1.1⁺ cells derived from wild-type, Zc3h12d–/–, and Regnase-1–/– (Zc3h12a^–/–) mice 3 h after the application of 10 ng/mL βactin-mRNA-CP-FITC and IL1β-mRNA-CP-FITC. The quantifications of the FITC signal in B220⁺CD11c⁺NK1.1⁺ cell nucleus are shown (left, n = 14, 17, 8, and 15 cells from wild-type and Zc3h12d–/– mice for βactin-RNA and IL1β-RNA, respectively. right, n = 7, 21, 7, and 21 cells from wild-type and Regnase-1–/– mice for βactin-RNA and IL1β-RNA, respectively; 15 z-stack images per cell). Ages of Regnase-1–/– mice were close to Zc3h12d–/– mice. b Representative immunostaining for phospho-H2AX in the nucleus of B220⁺CD11c⁺NK1.1⁺ cells 3 h after the uptake of IL1β-mRNA-CP (top). Quantitative analysis of phospho-H2AX in B220⁺CD11c⁺NK1.1⁺ cells derived from wild-type and Zc3h12d–/– mice 3 h after the uptake of βactin-mRNA-CP gapdh-mRNA-CP IL1β-mRNA-CP and IL1β-stop-mRNA-CP (n = 3 biologically independent samples). Bar, 5 μm. c Time course of secondary necrosis in B220⁺CD11c⁺NK1.1⁺ cells derived from TCM-stimulated wild-type and Zc3h12d–/– (Homo) mice in the application of 20 ng/mL RNA. βactin-mRNA-CP, IL1β-mRNA-CP, and IL1β-stop-mRNA-CP were depicted as actb, IL1b, and IL1b-stop, respectively. Real-time tracking of fluorescein-DNA dye indicates the loss of membrane integrity resulting in late-stage apoptosis. d Detection system of entrained ex-IL1β-mRNA bound to Pol II in ZC⁺RAW cells (left). Real-time PCR analysis of IL1β-chimera-mRNA associated with Pol II in the nucleus. Anti-luciferase (Luc) antibody is the control (n = 3 biologically independent samples). e qPCR-derived quantifications of IL1rn and Dusp1 expression in nuclear RNA of ZC⁺RAW after application of IL1β-chimera-mRNA (n = 5 and 6 biologically independent samples at 30 and 120 min, and n = 12 biologically independent samples at 30 and 120 min for IL1rn and Dusp1, respectively). f qPCR-derived quantifications of IL1rn and Dusp1 expression in B220⁺CD11c⁺NK1.1⁺ cells after the application of βactin-mRNA and IL1β-mRNA. Representative data are shown in the graph. Repeated experiment data are shown in Supplementary Fig. S9. In the graphs, averages ± SEM and the results of Student’s t-test (two-sided) or one-way ANOVA with Bonferroni correction are shown. The P values are shown in the figure. Source data are provided as a Source Data file.