Fig 2: Comparative analyses of pentapartite oral fluid (OF) fractions between healthy volunteers and patients with OSCC. (a) Western blot analyses of pentapartite fractions from six healthy subjects (HC7 to HC12) and six patients with OSCC (OSCC1 to OSCC6). Membranes were probed with antibodies against CD81, CD63, CD9, Alix, Hsp70, ß-actin, AQP5, ACTN4, CD133, EGFR, and E-cadherin. Numbers on the right indicate molecular weight (MW) × 10–3 of for MW markers. Black arrows show the reported sizes of the proteins. Nanoparticle tracking analyses (NTA) of particles contained in the 10K, 160K, and Sup fractions derived from healthy controls and patients with OSCC. Particle concentrations (b), and mode diameters (c), are indicated by green and red dots, respectively. The mean value of each group is represented as a solid horizontal line. The two-tailed unpaired t-test was used to evaluate statistical significance. Statistical significance was defined as p < 0.05. n.s. not significant.

Fig 3: Exosome characterization obtained by SEC. (A) ESP and protein concentration profiles across SEC fractions using NTA and microBCA analysis. (B) Particle size distribution of SEC fraction 4. (C) Western blot identification of exosome enriched proteins, Alix and CD63, from fraction 4 and (D) Gold immuno-EM analysis confirming presence of CD63 protein at the exosome membrane.

Fig 4: Distributions of protein markers, chromosomal DNA, and mitochondrial DNA in the pentapartite fractions of oral fluids (OFs) from 6 healthy volunteers. (a) Western blots of proteins markers for canonical exosomes (CD81, CD9, Alix, CD63 and HSP70), salivary gland (AQP5), mitochondria (ATP5A), chromatin (Histone H2B), and miRNA-associated protein (Ago2). Numbers on the right indicate molecular weight × 10–3. Black arrow shows the expected size of ATP5A. Asterisks in the 160K fractions of Ago2 are discussed in the text. Note that Sup was diluted 35-fold. The copy numbers of chromosomal DNA (b), and mitochondrial DNA (c), were determined by quantitative PCR. Target genes were SLCO2B1 and SERPINA1 for chromosomal DNA and ND1 and ND3 for mitochondrial DNA. The OFs were obtained from n = 6 healthy donors. Data are expressed as the mean ± standard deviation (SD). Dots indicate the mean value of the three measurements for each subject, while the mean values obtained from six subjects are represented as solid horizontal lines. Note that Sup was diluted 35-fold. The data obtained from HC45 are shown by red filled circles (see “Results” section).

Fig 5: Baseline characterization of RBC-extracellular vesicles (EVs) in transgenic murine model.(A) Schematic representation of the experimental murine models. Erythroid lineage specific conditional Cre knock-in mice (EpoR-Cre mice, BALB/c background) were crossed with membrane-targeted Tomato/membrane-targeted GFP (mTmGRosa26 mice, C57BL6/Sv129) to produce double transgenic (EpoR-Cre/mTmG) off-springs. Activated Cre mediates the removal of membrane-targeted tandem dimer Tomato (mT) sequence allowing the expression of membrane-targeted green fluorescent protein (mG) in RBCs. Arrows represent the direction of transcription. Triangles represent loxP sites for Cre-mediated recombination. pA stands for polyadenylation sequences. (B) High-magnification fluorescence images (63×) of fresh prepared peripheral blood smear and cell-free plasma smear from mTmG and EpoR-Cre/mTmG mice to characterize the mature circulating RBCs, and RBC-EVs (n = 4 in each group). Small bright GFP positive dots in the peripheral blood smear represent platelets. Scale bar: 10 µm. (C) Nanoflow cytometric characterization of cell-free plasma from mTmG mice and EpoR-Cre/mTmG mice, showing presence of mGFP+ RBC-EVs in EpoR-Cre/mTmG mouse plasma. The auto fluorescence from the Suspension Buffer (Phosphate Buffer Saline) has been accounted for, as background. Shown are Td on the y-axis and GFP in the x-axis and divided in four quadrants (Q1 is mTd+ [red]; Q2 is mTd+/mGFP+ [double positive]; Q3 is mGFP+ [green], and Q4 is background). (D) Real-time PCR analysis of Cre-recombinase mRNA in bone marrow, RBC-EVs and RBCs from EpoR-Cre mice. Data are shown as raw Ct values. n = 3 each. (E) Real-time PCR analysis of miRNA-451a in bone marrow, RBC-EVs and RBC from EpoR-Cre mice. Data are shown as raw Ct values. n = 3 each. (F) Western blots of RBC-EVs protein lysates from EpoR-Cre/mTmG and mTmG mice probed with the indicated antibodies: Cre-recombinase, Ter-119 (mature RBC- specific marker), and the exosome specific surface marker Alix. Data shown are representative of three replicated independent experiments. (G) Western blots of RBCs and bone marrow protein lysates probed with the indicated antibodies. Data shown are representative of three replicated independent experiments.