CD63 Antibody - #AF5117

Figure 2 Characterization of ESIONPs@EXO derived from ESIONPs engineered macrophages. (A) The morphology of EXO and ESIONPs@EXO determined by TEM. Scale bar: 200 nm (left) and 100 nm (right). (B) The size distribution of EXO and ESIONPs@EXO evaluated by NTA. (C) Western blot analysis of CD9, CD63, CD81, TSG101, and calnexin. (D) Relaxation properties of ESIONPs@EXO. (E) T1 and T2 weighted MR images of ESIONPs@EXO at different concentrations (measured on a 3 T MR scanner). 1/T1 (F) and 1/T2 (G) relaxation rates of ESIONPs@EXO at different concentrations.

Figure 2 TGF-β1 promotes the secretion of exosomes by renal tubular epithelial cells and activates fibroblasts in vitro. (A) Schematic diagram of experimental process. Exosomes from NRK-52E cells treated without (Ctrl-Exos) or with TGF-β1 (TGFβ1-Exos) were extracted and incubated with NRK-49F cells. (B,C) DLS and NTA of exosomes from NRK-52E cells. (D) TEM image of exosomes isolated from NRK-52E cells. Scale bar = 100 nm. (E, F) Representative western blot (E) and quantitative data (F) of CD63 as an exosome marker in exosomes from TGF-β1- or GW4869-treated NRK-52E cells. Numbers (1 to 3) indicate each independent treatment in the given group. *p < 0.05 versus Ctrl-Exos, #p < 0.05 versus 5 ng/ml TGFβ1-Exos, &p < 0.05 versus 15 ng/ml TGFβ1-Exos (n = 3). (G) Fluorescent staining image of PKH-67-labeled NRK-52E cells. Scales bars=50 μm. (H) Fluorescent staining image of NRK-52E cell-derived exosomes taken up by NRK-49F cells. Scales bars=10 μm. (I, K) Representative western blot (I) and quantitative data (K) of α-SMA and PCNA in NRK-49F cells incubated with exosomes from TGF-β1- or GW4869-treated NRK-52E cells. Numbers (1 to 3) indicate each independent treatment in the giving group. *p < 0.05 versus Ctrl-Exos, #p < 0.05 versus 5 ng/ml TGFβ1-Exos, &p < 0.05 versus 15 ng/ml TGFβ1-Exos (n = 3). (J) Proliferation rate of NRK-49F cells incubated with NRK-52E cell-derived exosomes measured by CCK-8. *p < 0.05 versus Ctrl-Exos, #p < 0.05 versus 5 ng/ml TGFβ1-Exos, &p < 0.05 versus 15 ng/mL TGFβ1-Exos (n = 3). (L-N) Double immunofluorescence staining (green for Col-I and red for fibronectin) demonstrates the expression of Col-I and fibronectin in NRK-49F cells incubated with NRK-52E cell-derived exosomes. Scales bars=50 μm. *p < 0.05 versus Ctrl-Exos, #p < 0.05 versus 5 ng/ml TGFβ1-Exos, &p < 0.05 versus 15 ng/mL TGFβ1-Exos.

Fig. 2. SMφ-derived EVs induce ferroptosis in pulmonary microvascular endothelial cells. (A) The flowchart of EV enrichment by ultracentrifugation. (B) Transmission electron microscopy (TEM) images of EVs isolated from the medium of Mφ and SMφ. Scale bar = 100 nm. (C) Representative nanoparticle tracking analysis images showing the distribution and average diameter of EVs. (D) Representative immunoblotting of EV markers. (E) Representative immunofluorescence staining of Hoechst/CFSE after co-culture with human pulmonary microvascular endothelial cells (HPMECs) using CFSE-labeled EVs. Scale bar = 10 μm. (F) Quantification of CCK8 assays performed on HPMECs co-cultured with Mφ-EVs and SMφ-EVs (n = 5). (G) Representative images and quantification of the Zombie NIR™ staining in HPMECs co-cultured with Mφ-EVs and SMφ-EVs in the presence or absence of ferrostatin-1 (Fer-1) (n = 3). (H) Representative immunofluorescence staining for DAPI/CD31/ZO-1 and DAPI/CD31/occludin in HPMECs. Scale bar = 50 μm. (I) Representative immunoblotting and quantification of barrier-associated proteins (ZO-1, VE-Cadherin and occludin) in HPMECs (n = 3). (J) Representative images of mitochondrial membrane potential of HPMECs. Scale bar = 50 μm. (K) Representative images of liperfluo staining in HPMECs. Scale bar = 50 μm. (L) C11-BODIPY assay evaluating the lipid ROS levels in HPMECs (n = 3). (M) Representative immunoblotting and quantification of SLC7A11 and GPX4 in HPMECs (n = 3). (N) Representative images of immunofluorescence staining for DAPI/CD31/SLC7A11 and DAPI/CD31/GPX4 in HPMECs. Scale bar = 50 μm. Data are presented as mean ± SD.

Fig. 3. Synthesis and identification of cEVs and cEVsingle bondB6. (A) Schematic overview of synthesis and collection of cEVs. (B) Shown are transmission electron microscopy (TEM) and size distribution of cEVs and cEVsingle bondB6. Scale bars, 100 μm (inset). (C, D) NTA profiles of cEVs (C) and cEV-B6 (D) under optimized storage conditions (4 °C, 5 days), confirming colloidal stability. (E, F) Zeta potential of cEVs (E) and cEV-B6 (F) stored at 4 °C over 5 days, demonstrating surface charge stability. (G) Western blot analysis of classic biomarkers on EVs secreted by CAFs, including β-tubulin, Epcam, TSG101 and CD63. (H) Time-dependent cellular uptake kinetics of DiL-labeled cEV-B6 in CAFs and LLC, quantified by flow cytometry. All data are expressed as means ± SD. n.s. means no significance.