Fig 1: Correlation between LDL size and ApoF binding in hyperlipidemic plasmas. The size of LDL in hyperlipidemic and normolipidemic plasmas was determined by gel filtration FPLC. A: Typical LDL elution profiles for LDL. B: Correlation between the size of LDL from normolipidemic, HyperTC, and HyperTG subjects, as determined by FPLC, and the amount of ApoF per LDL particle expressed as the ratio of ApoF/ApoB. Shown are the mean ± SEM of n = 4–5 plasma pools for each lipid group. Note that the x-axis is reversed so that LDL size increases from left to right.
Fig 2: Characterization of crude, LPP and EV extracts obtained by size and density‐based fractionation of blood plasma. (a) Graphical overview of the sequential biophysical separation approach including size‐exclusion chromatography (SEC) and OptiPrep density gradient (ODG) centrifugation. (b) Western blot (FLOT1) and ELISA analysis (CD9) of SEC fractions. (c) Western blot (APOA1) and ELISA analysis (APOA1, APOB) of SEC fractions. (d) Western blot (FLOT1), ELISA analysis (CD9) and protein concentration of ODG fractions. (e) Western blot (APOA1) and ELISA analysis (APOA1, APOB) of ODG fractions. (f) TEM analysis of crude (scale bar: 200 nm), LPP (scale bar: 500 nm) and EV extracts (scale bar: 200 nm (left and right)) and overview of the particle enrichment relative to proteins, and APOA1‐containing and APOB‐containing LPP. All SEC and ODG fractions were loaded in equal volumes for western blot analysis. Note the differential axis labelling in (b) (expressing CD9 in μg/mg) and (d) (expressing CD9 in μg/ml)
Fig 3: Immunoblotting for EV biomarkers and potential albumin contamination and ELISAs for potential ApoB contaminationEV biomarkers and albumin contamination in EVs from the traditional DIFF-UC method, n = 3 separations (A). Representative immunoblots following the other 5 methods i.e. controls (B) and BC (C), summarised in (D). ELISA of ApoB contamination of controls (E) and BC (F) EVs. Hs578Ts(i)8 cell lysate was the control. Graphs are mean of n = 3±SEM experiments. One-way ANOVA; *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 4: Small EVs vs. whole serum nanoparticles. (A) Comparison of nanoparticle concentration by size range. (B) ApoB quantification on small EVs and whole plasma in all samples. (C) ApoB quantification on small EVs and whole plasma in Pre- and Post-surgery samples. (D) ApoA1 quantification on small EVs and whole plasma in all samples. (E) ApoA1 quantification on small EVs and whole plasma in Pre- and Post-surgery samples. *p < 0.05.
Fig 5: Detecting CMs using TEM and ApoB ELISA. (a) Transmission electron microscopy (TEM) images show spherical particles ranging from 250 to 500 nm, characteristic of CMs. (b) Standard curve absorbance values were obtained from Apolipoprotein B (ApoB) ELISA standards (n = 2) and plotted with the concentration of ApoB on the x-axis linearly correlated with absorbance at 450 nm on the y-axis. (c) Concentration of ApoB-48 in isolated CM samples was consistent across three test compounds was quantified (n = 6 per compound). Statistical comparisons between groups using 1-way ANOVA (P > 0.05) indicated insignificant differences in ApoB levels (as a surrogate for CM levels) between different compounds. CAB = cabazitaxel. MET = metformin. TXL = tenofovir exalidex.
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