Composition of EVs markers under normoxic and hypoxic conditions depends on the expression level of adaptor protein Ruk/CIN85 in mouse renal carcinoma Renca cells

Abstract

Abstract Aim: To isolate and characterize extracellular vesicles (EVs) produced by mouse renal carcinoma Renca cells with different expression levels of the adaptor protein Ruk/CIN85 under normoxia and hypoxia conditions. Methods: The density gradient centrifugation was used to isolate EVs from the conditioned medium of Renca cells cultured under normoxia and hypoxia conditions. Further characterization of EVs was performed by using nanoparticle tracking analysis (NTA), electron microscopy and Western Blot analysis. Results: Significant differences in average particle size between EVs produced by sublines studied under experimental conditions were not found. At the same time, concentration of particles produced by Ruk/CIN85 overexpressing cells turned out to be an order of magnitude higher in hypoxia in comparison to normoxia conditions. It was shown that under normoxia conditions the content of both Ruk/CIN85 and EVs’ markers Alix and CD81 was increased in vesicles produced by Renca cells with Ruk/CIN85 overexpression in comparison with those from control mock-transfected cells. Under hypoxia conditions, the content of studied proteins decreased by more than two orders of magnitude in EVs secreted by Renca cells with up-regulation of adaptor protein whereas the content of Ruk/CIN85 and CD81 increased in EVs from mock-transfected cells. Conclusions: It has been demonstrated that the adaptor protein Ruk/CIN85 is a novel component of EVs produced by tumor cells that may play a role in the control of EV composition under normoxia and hypoxia