Fig 2: (A) NTA analysis of the size distribution of L1CAM-immunoprecipitated NDEVs. (B) Immunoblots of Alix, CD9, ApoA1, and GM130 in total EVs and NDEV samples. * Nonspecific band.

Fig 3: Characterization of EVs from breast cancer cells cultured at each temperature. (a) Size distribution of EVs from cancer cells cultured at different temperatures. The sizes of EVs from cancer cells cultured at 35°C, 37°C, or 39°C were analyzed using a NanoSight nanoparticle tracking system. The vertical and the horizontal axes in the graphs indicate the number of EV particles (×106)/ml and particle size (nm), respectively. Numbers (red colour) shown on graphs are the average sizes of the EVs. (b) Electron microscopy analysis of EVs. Representative images of EVs from MDA-MB-231 cells cultured at 35°C, 37°C, or 39°C. Scale bar represents 100 nm. (c) Western blot for CD63, CD9, ALIX, and APOA1 of EVs from MDA-MB-231 cells cultured at 35°C, 37°C, or 39°C

Fig 4: Western blot analysis of L1CAM, Alix, ApoA1 and CD9 in L1CAM + NDEVs, Total EVs, and EV free plasma, N = 3 cognitively normal individuals (A). Representative size distribution of three NDEV samples by NTA analysis. Average number of NDEVs was 2.73 × 1010 ± 1.72 × 1010 particles/mL and the mode of NDEV diameters was 179.44 nm ± 51.59 nm (B). Representative negative stain (Oxalate) Transmission Electron Microscopy images of NDEV samples (C). Exoview analysis of NDEVs by using ExoView Tetraspanin chip and characteristic image; the analysis demonstrates that NDEVs express all major tetraspanins and that a majority of them express CD9 compared to CD81 and CD63 (D).

Fig 5: Evaluation of host biomarkers for Dutch COVID-19 patients and healthy controlsLevels of IL-6, IP-10, ferritin, SAA1/A2, CRP, ApoA1, and S100A12 were measured by UCP-LFA in serum samples of COVID-19 patients (n = 102) and healthy controls (n = 39; n = 27 from before (May) 2019 (n = 12 from after 2019 (June/July 2020)) from the Netherlands. Median values for each group are indicated by horizontal bars. Mann-Whitney U tests were performed to determine the statistical significance between groups (pvalues: *p=0 · 05, **p=0 · 01, ***p=0 · 001, ****p=0 · 0001). Orange dots: healthy controls from before 2019; purple dots: healthy controls from after 2019; black dots: COVID-19 patients with a fatal outcome; yellow dots: COVID-19 patients with severe disease; turquoise dots: COVID-19 patients with moderate disease. AUC: area under the curve; COVID-19: coronavirus disease 2019; Fc: flow control line; T: test line.