Fig. 4.

Extracellular vesicle induced transcription of NF-κB. a EVs were isolated from HCT116 cells. EVs were resuspended in PBS and co-cultured with non-malignant 1459 cells and malignant HCT116 cells. After 7 days of co-culture, cells were transfected with NF-κB reporter plasmid to measure NF-κB transcriptional activation [16]. After 24 h, the samples were harvested, washed twice, lysed and combined with a luciferase assay reporter. The data is reported as the mean +/- s.d. of 2 independent experiments performed in triplicate. Relative to 1459, HCT116 cells showed increased activity of NF-κB transcription, *p < 0.00001. Increased relative activity of NF-κB transcription was also seen in 1459 cells co-cultured with HCT116EVs, relative to 1459 control, **p = 0.00004. b EVs were isolated from HCT116 cells and NF-κB transcriptional reporterassay was performed as described in a in the presence or absence of 3 μm BAY, a irreversible inhibitor of NF-κB [30]. Relative to 1459 control, 1459 cells in the presence of 3 μm BAY showed decreased activity of NF-κB transcription, *p = 0.025. Increased relative activity of NF-κB transcription was observed in 1459 cells co-cultured with HCT116EVs, relative to 1459 control, **p < 0.0001. In the presence of 3 μm BAY, 1459 + HCT116 EV cells showed decreased activity of NF-κB transcription, as compared to those in the absence of BAY, ***p < 0.0001. c Cell migration assay was performed using 1459 cells co-cultured with HCT116 EV in the presence or absence of 3 μm BAY [30]. Relative to 1459 control, 1459 + BAY cells showed decreased cellular migration, *p = 0.047, as assessed by paired t-test. An increase in cellular migration was assessed in 1459+ HCT116 EV co-cultured cells, relative to 1459 control, **p < 0.0001. Co-culture of 1459 cells with EVs from HCT116 cells treated with 3 μM BAY showed decreased cellular migration, ***p < 0.0001, compared to 1459 + HCT116 EV co-cultured cells. The assay was performed twice in triplicate