Figure 5

EV-RNA contributes to NSCLC EV-mediated phenotypes. (A) Representative images showing the invasive effect of RNase A treated Calu6-EVs on BEAS-2B cells (Scale bar, 200 μm). The number of invasive cells was compared between treatments using unpaired t-test with Welch’s correction. There was no significant difference between BEAS-2Bs incubated with and without RNase A treated Calu6 EVs. Untreated sample is represented as UT (B) Correlation between EV protein (µg) and EV-RNA (ng) indicates a strong linear correlation with an R² = 0.64, the dotted line represents the 95% confidence interval for the linear correlation. (C) EV-RNA yield per million EVs is not statistically different between tumorigenic (Calu6 and H358) and non-tumorigenic (BEAS-2B) cells. (D) Representative images showing the invasive effect of EV-RNA on BEAS-2B cells (Scale bar, 200 μm). EV-RNA concentrations relative to 1, 5, and 10 µg/mL of EV-protein were calculated (see Table S1). BEAS-2B cells were transfected with the indicated protein-equivalent EV-RNA concentration and invasion was quantified 48 h following transfection. EV-RNA from NSCLC EVs (Calu6 and H358) significantly increase the number of invasive cells in comparison to premiR-NC. p-values were determined using one-way ANOVA followed by Dunnett’s multiple comparison tests (n = 3, **p < 0.01, ***p < 0.0005 and ****p < 0.0001). (E) Representative histograms demonstrate the levels of surface E-cadherin on BEAS-2B cells following transfection with negative control RNA (siNC2 or premiR-NC) or Calu6 EV-RNA relative to 300 µg/mL of EV-protein (n = 2).