Fig. 4: Analysis of cellular pathways to study EV-mediated RNA transfer and uptake. | Nature Communications

Fig. 4: Analysis of cellular pathways to study EV-mediated RNA transfer and uptake.

From: A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA

Fig. 4

a Workflow to study the role of specific genetic targets in intercellular RNA transfer using the CROSS-FIRE system. b Flow cytometry analysis of HEK293T Stoplight+spCas9+ reporter cells after a five day co-culture with sgRNA+ MDA-MB-231 donor cells subjected to siRNA-mediated KD of genes as compared to a non-targeting negative control siRNA (NC). Means + SD, n = 5 independent experiments, Dunnett’s multiple comparison test. c Flow cytometry analysis of HEK293T Stoplight+spCas9+ reporter cells after a five day co-culture with sgRNA+ MDA-MB-231 donor cells, in which HEK293T reporter cells were subjected to siRNA-mediated KD of various genes as compared to a non-coding control siRNA (NC). Means + SD, n = 4 independent experiments, Dunnett’s multiple comparison test. d Schematic of workflow to study the role of genes involved in EV-mediated RNA delivery. e Flow cytometry analysis of Stoplight+spCas9+ HEK293T reporter cells treated for six consecutive days with sgRNA+ MDA-MB-231-derived EVs after transfection with a non-coding siRNA (NC), or siRNAs targeting Cav1, as compared to vehicle-treated reporter cells (PBS). EVs were added every 24 h for six additions with an average concentration of 1.8e11 ± 1.1e11 EVs per addition. Means + SD, n = 3 biological replicates, Tukey’s multiple comparison test. f Flow cytometry analysis of HEK293T Stoplight+spCas9+ reporter cells after a five day co-culture with sgRNA+ MDA-MB-231 donor cells, in which HEK293T reporter cells were subjected to siRNA-mediated KD of various novel genes as compared to a non-coding control siRNA (NC). Means + SD, n = 4 independent experiments, Dunnett’s multiple comparison test. g Flow cytometry analysis of HEK293T reporter cells after a five day direct co-culture experiment with sgRNA+MDA-MB-231 donor cells with or without the presence of ROCK-inhibitor Y27632 at a concentration of 1 μM. Means + SD, n = 3 biologically independent samples, Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.

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