Fig. 4: Small extracellular vesicle-mediated transport of amiR-4 to uninfected cells contributes to enhanced cytotoxicity.

a Immunoblotting analysis of ALIX, CD9 and TSG101 in total purified SEVs produced by indicated cell lines with or without infection with MRB or MRBΔG (n = 3). b NTA showing size distribution and quantification of SEVs produced from mock-infected or MRB∆G-infected 786-0 cells (n = 3). c RT-qPCR analysis of amiR-4 levels compared to hsa-Let-7f-5p (loading control) in mock cell-associated SEVs and infected cell-associated SEVs derived from 786-0 cells infected with MRB∆G-amiR-4. Results displayed as mean ± SD, n = 3. ND   not detected. d Representative immunofluorescence images showing uptake of CFSE-labelled SEVs derived from MRBΔG-amiR-4-infected 786-0 cells by naive 786-0 cells (n = 2). Nuclei were stained with Hoechst dye. Scale bar = 10 μm. e, f Indicated cancer cells were educated with SEVs harvested from mock-infected, MRB∆G-amiR-NTC or MRB∆G-amiR-4-infected cells and treated with vehicle control (DMSO) or GSK126. Representative crystal violet cell cytotoxicity assay images (e) and their corresponding cell viability quantification compared to mock SEV and DMSO-treated cells (f) are shown; the timeline of treatment procedures is shown in Supplementary Fig. 4g. Shown are means ± SD, n = 3. Two-way ANOVA with Sidak’s multiple comparisons test (95% CI), ****P < 0.0001. g Schematic representation of transwell coculture assays designed to assess the transfer of amiR-4 via infected cell-derived SEVs to uninfected cells. h RT-qPCR analysis of amiR-4 levels in receiving 4T1 wild-type (WT) cells (lower compartment) after 48 h of education by cell-secreted factors derived from MRB∆G-amiR-4-infected cells (4T1 wild-type [WT] or Rab27a-deficient [Rab27a^−/−] cells; upper compartment). Results displayed as mean ± SD, n = 3, two-tailed unpaired t test, **P = 0.0066. i, j 4T1 WT (n = 4) or Rab27a^−/− cells (n = 7) were mock-treated or infected with VSVΔ51 control or VSVΔ51-amiR-4 (MOI 0.025) and treated with either vehicle control (DMSO) or GSK126 (15 μM). Representative crystal violet cell cytotoxicity assay images (i) and their corresponding quantifications (j) are shown as mean ± SEM, two-way ANOVA with Tukey’s multiple comparison test (95% CI), ^nsP > 0.05, ***P = 0.0003. Source data are provided as a Source data file.