Fig. 3: ZC3H12D brings nex-IL1β-mRNA into the nucleus.

a Fluorescence confocal microscopy of ZC⁺RAW after application of 10 ng/mL non-labeled IL1β-mRNA, FITC-labeled βactin-mRNA, and IL1β-mRNA with or without Cap-Poly(A) (CP). 3D images are shown (left). Quantitative analysis of RNA-FITC in the nucleus (right). IL1β-stop-mRNA has three stop codon mutations in the CDS (top). The non-labeled IL1β-mRNA was used as a basal control for the FITC signal (n = 5, 5, 5, 4, and 5 cells for non-labeled IL1β-RNA, FITC-labeled βactin-RNA, gapdh-RNA, IL1β-RNA, and IL1β-stop-RNA respectively). Repeated experiment data are shown in Supplementary Fig. S9. b System for the detection of external IL1β-mRNA using human-mouse chimera IL1β-mRNA (IL1β-chimera) followed by reverse transcription with a 3′-mGSP and a 5′-human probe in the RT-qPCR analysis (top; more details in Supplementary Fig. S3b). Detection of IL1β-chimeric-RNA entry into ZC⁺RAW. Nuclear RNA and cytoplasmic Hotair and βactin normalized nuclear RNA and cytoplasmic RNA, respectively (n = 7, 8, 8, 8, and 8 biologically independent samples for 0, 30, and 120 min with IL1β-chimera, 30 and 120 min with IL1β-chimera-CP, respectively). c 3D images showing IL1β-mRNA-FITC and IL1β-stop-mRNA-FITC uptake in the nucleus of B220⁺CD11c⁺NK1.1⁺ cells derived from TCM-stimulated wild-type and Zc3h12d–/– mice 3 h after the application of 10 ng/mL RNA (top). Quantitative analysis of RNA-FITC in the nucleus of B220⁺CD11c⁺NK1.1⁺ cells from wild-type mice (bottom left, n = 6, 4, 6, 6, and 6 cells for non-labeled IL1β-RNA, FITC-labeled βactin-RNA, gapdh-RNA, IL1β-RNA, and IL1β-stop-RNA respectively) and from Zc3h12d–/– mice (bottom right, n = 6, 6, 6, 6, and 5 cells for non-labeled IL1β-RNA, FITC-labeled βactin-RNA, gapdh-RNA, IL1β-RNA, and IL1β-stop-RNA respectively). d Fluorescence confocal microscopy of ZC⁺RAW after the addition of FITC-labeled βactin-mRNA, full length, CDS, or 3′-UTR of IL1β-mRNA (top). Quantitative analysis of 10 ng/mL IL1β-mRNA domain-dependent nucleus uptake (bottom; n = 10, 10, 11, 10, and 10 cells for non-labeled IL1β-RNA, FITC-labeled βactin-RNA, full length, CDS, and 3’UTR of IL1β-RNA, respectively). In this analysis, 15-stacked 3D images were obtained from the bottom to the top of the nucleus. Bar, 5 μm. In the graphs, averages ± SEM and the results of 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.