Fig 2: Relative expression of tetraspanins (a–e); Caveolin-1 (f); Ezrin (g); Moesin (h); Radixin (i); and Alix (j) in thyroid cell lines. Data are reported as means with standard deviation (±SEM). *: p < 0.05; ** p < 0.01; ***: p < 0.001.

Fig 3: Cell adhesion is dispensable for the recruitment of the MKLP1 and CEP55 but required for the ESCRT recruitment to the midbody(A, B, F) Representative immunofluorescence micrographs illustrating the distribution of selected proteins in the F/M region (labeled with Aurora B) at the beginning of cytokinesis and after 60 minutes incubation of BJ cells adhering to fibronectin or kept in suspension. (A) MKLP1 (green, upper left panel) and CEP55 (green, upper right panel), (B) TSG101 (green, lower left panel) and ALIX (red, lower right panel), (F) CHMP4B (green). The square frames show the midbody area at higher magnification. Arrows indicate midbody regions in the merged pictures. Nuclei were stained with DAPI (blue). Mean % ± SD of the number of mitotic cells in cytokinesis lacking (C) MKLP1 and CEP55, (D) TSG101 and ALIX and (G) CHMP4B in the F/M regions. ***, **, and * represent P-value less than 0.001, 0.01 and 0.05, respectively. (E) Representative western blot of cell lysates illustrating the level of ALIX and TSG101 in exponentially proliferating cells (Control) and synchronized mitotic cells directly after isolation (Zero) and following two hours incubation under the adhesion or suspension conditions.

Fig 4: Possible secretome-mediated immunomodulation by MSCs. (a) Experimental design for the intraperitoneal injection of anti-PD-L1 monoclonal antibody (mAb) and intravenous injection of PKH-labelled hMSCs in NOD mice. The tissues were dissected 24 h after the final injection. (b) Fluorescence of PKH and DAPI in the lung, pancreas, liver and spleen. Scale bars, 100 µm. (c) Experimental design for the intraperitoneal injection of anti-PD-L1 mAb and intravenous injection of hMSCs in NOD mice. The blood plasma was collected at 4 h after final injections [control: mean blood glucose level was 7.5 mmol/l; MSC(-): mean blood glucose level was 11.6 mmol/l; MSC(+): mean blood glucose level was 7.6 mmol/l; those exceeding the upper limit of measurement with a simple blood glucose meter were regarded as having a blood glucose level of 33.3 mmol/l]. (d–h) Plasma extracellular vesicles (EVs) were purified, and the amounts of exosome marker proteins were evaluated by western blotting. Exosome markers: Alix and syntenin by human and mouse species unspecific antibodies; CD63 and MFG-E8 by human-specific antibodies (d). (e–h) Comparison of the plasma exosome levels between groups by exosome markers in EV-fractions: Alix (e), hCD63 (f), hMFG-E8 (g) and syntenin (h) (all groups n=6). (e–h) Data are shown as the mean ± SEM. *p?0.05; **p<0.01; ***p?0.001 between the indicated two groups by Student’s t test. (i–k) Plasma cytokine profile was determined in cytokine arrays using pooled plasma (n=6) of the MSC(-) group (i) and the MSC(+) group (j). Cytokine profiles were compared between groups (k). hAD-MSCs, human adipose-derived mesenchymal stem cells; hCD63, human CD63; hMFG-E8, human milk fat globule-EGF factor 8 protein

Fig 5: The induction of a CAF phenotype by GOF p53 from cancer cells(A) Characterizations of small EVs from HT29-ntsh and HT29-shP53 cells using NTA and TEM assays. Scale bar, 100 nm.(B) The p53 knockdown efficiency in HT29 cells and the p53 expression in relatively small EVs. Pixel densities for p53 bands in comparison with GAPDH and CD63 bands normalized to 1.0 are shown. CD63, Alix, and TSG101 were positive markers for small EVs, whereas GRP94 was a negative marker for them. Data are represented as mean ± SD. *p < 0.05; **p < 0.01.(C) Volcano plot of differentially enriched variables in small EVs representing the logarithm of fold change (log2) on the x axis and -log10 of the p value on the y axis.(D) Distribution of different types of RNAs in ntsh-sEVs and shP53-sEVs.(E) The frequently enriched upregulated pathways, interaction pathways, and the upstream regulators in NoF 151 fibroblasts following treatment with GOF p53-containing small EVs.(F) FAP, IL1B, TGFB, and CXCL12 mRNA expression levels in NoF 151 fibroblasts after treatment with ntsh-sEVs and shP53-sEVs in the presence or absence of Nrf2 inhibitor (Nrf2i) ML385. Data are represented as mean ± SD. *p < 0.05; **p < 0.01; n.s., not significant.(G) Results of a human cytokine array performed using supernatants of NoF 151 fibroblasts after treatment with ntsh-sEVs or shP53-sEVs.See also Figure S3 and Table S2. Raw data shown in Data S1.