fig7

Figure 7. Functional transfer of miR-100 and miR-125b. (A) RNA was collected from EVs from CC and CC-CR cells and the levels of miR-100 and miR-125b were quantified by RT-qPCR. The left panel shows the fold-change enrichment of these miRNAs in EVs compared to cellular levels. The right panel shows the fold change in EV levels of miR-100 and miR-125b when comparing CC-CR and CC cells; (B) Transwell co-culture experiments were performed with ΔmiR-100/miR-125b recipient cells and the donor cells as indicated on the X-axis. RNA was collected from the recipient cells after 5 days in culture and fold changes in miR-100 and miR-125b levels were determined by RT-qPCR; (C) Schematic of Transwell co-culture experiments to test silencing of CGN and PTPRR luciferase reporter constructs in miR-100/miR125b recipient cells; (D) ΔmiR-100/miR-125b recipient cells were transfected with luciferase reporters and co-cultured with either ΔmiR-100/miR-125b (grey) or CC-CR (black) donor cells. Relative luciferase values (y-axis) were calculated in recipient cells, normalizing as in Figure 3. For (A and B), data (mean ± SEM, n = 3) were analyzed using one-way ANOVA tests while the data in panel (D) (mean ± SEM, n = 3) were analyzed using two-way ANOVA tests. For significance, ^* indicates P < 0.05, ^** indicates P < 0.01, ^*** indicates P < 0.001, and ^**** indicates P < 0.0001; ns is not significant. EVs: Extracellular vesicles; CC: cystic colonies; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; CGN: cingulin; PTPRR: protein tyrosine phosphatase receptor type-R; SEM: scanning electron microscopy; ANOVA: analysis of variance.