Fig. 1: Detection of ZC3H12D on the cell surface and tumor-associated nex-IL1β-mRNA.

a Summary of the RNA uptake model in which ZC3H12D captures and transfers nex-mRNA to the nucleus. b FACS analysis of surface and intracellular ZC3H12D protein in PBMCs derived from mice without tumors or tumor-bearing mice. Several anti-ZC3H12D antibodies (bold line No. 1 in Fig. 1 and Nos. 2 and 3 in Supplementary Fig. S1d) and a control isotype antibody (dashed line) were used. c Kinetics of ZC3H12D expression in splenic leukocytes after stimulation by TCM. Bar, 5 μm. Experiments were repeated twice with similar results. Validation of anti-ZC3H12D antibody is shown in Supplementary Fig. S10 (validation and gating strategy). d Flow cytometric analysis of ZC3H12D on the surface of splenic leukocytes derived from wild-type and Zc3h12d–/– mice 3 h after stimulation of TCM treated with DNase or RNase. NoCM, control CM without tumor cells. The addition of RNase A to TCM did not cause intracellular entry of ZC3H12D (TCM + RNase). Reduction of surface ZC3H12D after application of purified RNA from TCM (RNA-TCM). ZC3H12D signals in Zc3h12d–/– mouse leukocytes were due to nonspecific antibody binding. e Detection system of IL1β-mRNA using two RT primers and a TaqMan qPCR probe (top). Real-time PCR analysis of IL1β-mRNA in lung CM cultured with various TCMs, such as LLC-TCM, E0771-TCM, and B16-TCM, presented as LTCM, ETCM, and BTCM, respectively (n = 8, 8, 7, and 8 biologically independent samples for NoCM, LTCM, ETCM, and BTCM, respectively). Faint detection of IL1β-mRNA in liver CM cultured with various TCMs (n = 9 biologically independent samples). f Scheme of RNA isolation from TCMs and CM of lung EC. qPCR detection of hIL1β-mRNA in TCMs after incubation of human breast cancer cells, including MCF7, MDAMB231, T47D, and HCC1954 (bottom left, n = 3, 5, 3, 3, and 5 biologically independent samples for no, MCF7, T47D, HCC1954, and MDAMB231, respectively). Secretion of hIL1β-mRNA from human lung ECs stimulated by various TCMs (bottom right, n = 4, 3, 6, 6, and 8 biologically independent samples for no, MCF7, T47D, HCC1954, and MDAMB231, respectively). “No,” zero cancer cells. g Depiction of extraexosome and exosome RNA isolation. h Ratio of nonvesicular RNA (extraexosome RNA) compared to exosome RNA in MDAMB231-TCM. i Ratio of extraexosome RNA compared to exosome RNA in mouse lung culture medium with control CM or TCM. j qPCR analysis after direct trap of nonvesicular mRNA by ZC3H12D protein beads. Representative data are shown in the graph. Repeated experiment data related to d and h–j are shown in Supplementary Fig. S9. In the graphs, averages ± SEM and the results of Student’s t-test or one-way ANOVAs are shown. The P values are shown in the figure. Source data are provided as a Source Data file.