Fig. 8

hnRNPA2B1 mediates the packaging of circEHD2 into EVs. A, The expression level of circEHD2 in serum EVs from RCC patients based on the metastasis status. B, qRT-PCR analysis of the expression level of EVs-circEHD2 in OSRC-2, 786-O, and HK2 cells. C, The expression level of EVs-circEHD2 in RCC cells after knockdown of circEHD2. D, The expression level of EVs-circEHD2 in RCC cells transfected with circEHD2. E and F, Transmission electron microscopy (TEM) and NanoSight were used to characterize the purified EVs from OSRC-2 cells. Scale bars: 200 nm. G, Western blot analysis of EVs markers from OSRC-2 EVs or cell lysates. H, qRT-PCR analysis of the expression level of circEHD2 in EVs from RCC cells treatment with GW4869 (an inhibitor of EVs secretion). I, qRT-PCR analysis of the expression level of circEHD2 in the CM of RCC cells after depletion of EVs by ultracentrifugation. J, Mass spectrometry (MS) analysis of the proteins interacting with circEHD2 via RNA pull-down assay. K, Western blot assay confirming the interaction between circEHD2 and hnRNPA2B1 in circEHD2 pull-down proteins. L, RIP assay in OSRC-2 cells confirmed that circEHD2 could be enriched by hnRNPA2B1. M, The three-dimensional structure showed that circEHD2 could interact with hnRNPA2B1. N, Subcellular co-localization of circEHD2 and hnRNPA2B1 in RCC cells measured by fluorescence staining assay. Scale bars: 10 μm. O, qRT-PCR analysis the expression level of EVs-circEHD2 after knockdown of hnRNPA2B1 in RCC cells. Error bars represent the standard deviation (SD) of three independent experiments. **, P < 0.01; ***, P < 0.001