Fig 2: Characterization of EVs: (A,B) particle size vs. percent intensity graphs of healthy donor (A) and COVID-19 (+) patients (B) SEVs/LEVs. (C) Mean of n = 5 sample size graph is mentioned. (D) TEM images of healthy donor and COVID-19 (+) patients SEVs and LEVs (representative image). An n = 5 each of healthy and COVID-19 SEVs and LEVs was used for particle size and TEM analysis. (E) Western blotting analysis of EV specific tetraspanin markers (CD63 and CD81) of healthy donor SEVs/LEVs and COVID-19 (+) SEVs/LEVs and EDP as negative control.

Fig 3: TPX2 can be transferred intercellularly through exosomes. (A and B) Transmission electron microscopy (TEM) images of exosomes isolated from A549 cells (Vector: A, TPX2: (B). Red arrows indicate exosomes. Scale bar: 200 µm. (C and D) The size distribution of exosomes determined from A549 cells (Vector: A, TPX2: (B) by nanoparticle tracking analysis (NTA). (E) Western blot analysis of exosome markers (tsg101 and CD63) in equivalent amounts of protein from A549-derived exosomes and A549 whole cell lysates (WCL) (as a control). (F) The amount of TPX2 mRNA in exosomes isolated from cell supernatant was detected by RT‒qPCR. (G and H) Western blot analysis of TPX2 expression in A549 (G) and H1299 (H) cells after incubation with exosomes. Data were statistically analyzed with paired two-tailed students, t-test. **p<0.01.

Fig 4: Optimization of polarization conditions for M2 and characterization of M2-Exos. A. Q-PCR identification of Arg1 and Mrc1 at different IL-4 concentrations of action for 24 h. B. Q-PCR identification of Arg1 and Mrc1 at different IL-4 (20 ng/ml) times of action. C. Western blot analysis of the surface markers of the exosomes: CD63, CD9, and CD81. D. TEM micrograph of M0-Exo and M2-Exo, scale bar: 100 nm. E. NanoSight detection of the size distribution of M0-Exo and M2-Exo.

Fig 5: miR-92a-2-5p and miR-373-3p are upregulated in myeloma cell exosomes and promotes bone destruction. (A) Dynamic light scattering (DLS) results for the size distribution and electron microscopic image of exosomes derived from culture medium of patient myeloma cells. Scale bar, 100 nm. Red arrows: Exosome. (B) Western blot analysis for HSP90, CD63, Calreticulin and β-actin in patient myeloma cells and exosomes. (C, D) Specific surface marker proteins CD9 and CD63 of exosome detected by flow cytometry. Precursors of osteoclasts were cultured in osteoclast medium treated with exosomes (20 µg/ml) isolated from MM.1S culture medium (MM.1S-exo), RPMI8226 culture medium (RPMI-exo) or patient myeloma cells culture medium (Pt MM-exo). Shown are the morphologies of multinuclear (≥ 3) TRAP+ cells (E) and TRAP 5b levels in cell culture supernatant (F). Scale bars, 100 µm. MSCs were cultured in osteoblast medium treated with MM.1S-exo, RPMI-exo or Pt MM-exo. Shown are representative images of Alizarin red S staining (G) and ALP activity (H) in MSCs. (I, J) Uptake of the red fluorescence dye Dil-labeled MM.1S-exo or RPMI-exo by precursors of osteoclasts or MSCs. Scale bar, 10 µm. Precursors of osteoclasts or MSCs were cultured in osteoclast medium or osteoblast medium treated with exosomes isolated from MM.1S (Vec and A2B1) or RPMI8226 (shCtrl and shA2B1) culture medium. Shown are numbers of multinuclear (≥ 3) TRAP+ cells (K) and summarized data of Alizarin red S staining (L). Addition of PBS served as a control. (M, N) MM.1S (Vec and A2B1) cells were cultured with or without GW4869 (20 µM) for 2 days, given fresh medium, and cultured for another 2 days, and then the conditioned medium (CM) was collected. Precursors of osteoclasts or MSCs were cultured with or without the MM.1S CM. After culturing, the cells were subjected to Alizarin red-S staining (M) or TRAP staining assay (N). ns, not significant. (O) Quantitative real-time PCR analysis shows the relative expression of miRNAs in patient myeloma cells with (n = 5) or without (n = 5) bone lesions. (P, Q) Quantitative real-time PCR analysis shows the relative expression of miR-92a-2-5p (P) and miR-373-3p (Q) in patient myeloma cells with (n = 10) or without (n = 10) bone lesions. (R, S) Correlation coefficient between the levels of miR-92a-2-5p (R) or miR-373-3p (S) and numbers of bone lesion in myeloma patients (n = 30). The correlations were evaluated using Pearson coefficient. r, correlation coefficient. P value was determined by Pearson coefficient. (T, U) Quantitative real-time PCR analysis shows the relative expression of miR-92a-2-5p (T) and miR-373-3p (U) in precursors of osteoclasts or MSCs treated with RPMI-exo or MM.1S-exo. (V, W) Shown are numbers of multinuclear (≥ 3) TRAP+ cells (V) and TRAP 5b (W) in precursors of osteoclasts infected with mimics of miR-92a-2-5p. (X, Y) Shown are summarized data of Alizarin red S staining (X) and ALP activity (Y) in MSCs infected with mimics of miR-373-3p. Data are averages ± SD. Each experiment was repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P values of P, Q, V-Y were determined by Student's t test. P values of F, H, K, L, M, N, T and U were determined using one way ANOVA.