Fig. 5

siRNA mediated Knock Down of 14-3-3 zeta/delta reduces soft agar colony formation. a HCT116 cells were transfected with siRNA to 14-3-3 zeta/delta, or equivalent amount of control siRNA solution. After 48 h incubation with siRNA to 14-3-3 zeta/delta, or equivalent amount of control, HCT116 cells were harvested for soft agar cloning and saved for Western Blot analysis. EVs were harvested from the HCT116 cells transfected with siRNA to 14-3-3 zeta/delta. EVs were resuspended in PBS and co-cultured with 1459 cells. After 7 days of co-culture, soft agar coloning was performed. There were 5 dishes/condition. The data represents the mean +/− s.d. of 3 independent experiments performed in quadruplet. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 transfected with 14-3-3 zeta/delta siRNA, compared to HCT116 transfected with scrambled siRNA as control, *p < 0.00001. Decrease in colony formation of 1459 + HCT116 14-3-3siRNA EV when compared to 1459 + HCT116 scramble siRNA EV was assessed, ** p = 0.0039. b After co-culture, cells were harvested and a portion of the cells were prepared as whole cell lysates for Western blot analysis. Western blot analysis confirmed the reduced expression of 14-3-3 zeta/delta in HCT116 + 14-3-3siRNA compared to HCT116 and HCT116 + scramble siRNA controls. Reduced expression of 14-3-3 zeta/delta was also seen in the co-culture of 1459 + HCT116 14-3-3 siRNA EV as compared to 1459 + HCT116 EV and 1459 + HCT116 scramble siRNA EV controls. c Western blot analysis of 14-3-3 sigma and epsilon protein levels of 1459 cells after co-culture with HCT116 EVs. d Western blot of 14-3-3 zeta/delta protein from vesicles isolated from 3 differential centrifugation preparations. MV = microvesicle fraction; EX = exosome fraction; T = MV and EX fraction