Figure 3.

Reducing DUSP11 increases 7SL RNA 5′-triphosphate levels in tumor-fibroblast extracellular vesicles. (A) Schematic diagram of the coculture conditioned media transfer assay. EV-containing conditioned media was concentrated from coculture media by differential centrifugation and transferred to MDA-MB-231 breast cancer (BrCa) cells silenced for DUSP11 or RIG-I. (B) RT-qPCR analysis of IFNB1 mRNA in siRNA-treated MDA-MB-231 cells incubated in concentrated coculture conditioned media as in A. Results are presented relative to those of untreated mock MDA-MB-231 cells. (C) Schematic diagram of the 5′ end characterization RT-qPCR assay of 7SL RNA isolated from coculture EVs. EV-containing pellets were collected from coculture conditioned media by differential centrifugation. Extracted RNA was treated with DNase I followed by treatment with or without 5′-monophosphate-specific exonuclease Terminator. (D) RT-qPCR analysis of 7SL RNA in EV RNA extracted from siNC or siDUSP11 (siD11) cocultured conditioned media. Results are presented relative to siNC coculture with 5S rRNA as the endogenous control. (E) RT-qPCR analysis of 7SL RNA in EV RNA isolated from siNC or siDUSP11 (siD11) cocultured conditioned media, treated with or without Terminator. Results are presented relative to mock-treated siNC coculture with 5S rRNA as endogenous control. (F) RT-qPCR analysis of 5.8S rRNA in EV RNA isolated from siNC or siDUSP11 (siD11) cocultured conditioned media, treated with or without Terminator. Results are presented relative to mock-treated siNC coculture with 5S rRNA as endogenous control. Data are derived from n = 3 independent replicates in B and D–F. In all panels, data are presented as mean ± SEM. (*) P < 0.05 (two-tailed Student's t-test).