CD9 Antibody - #AF5139

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 Preparation and characterization of ExoIRES-IL-10. (A) Schematic of the exosomes preparation and isolation process. (B) Western blot analysis of the exosomal inclusive markers (TSG101, CD9), exclusive marker (GM130), negative markers to confirm the purity (APOA1) in the isolated exosomes and parental cells. HEK293T cells were treated with PBS or transfected, empty vector, or IRES-Il-10 plasmids. (C) Representative transmission electron microscope (TEM) images of ExoNone, ExoEmpty or ExoIRES-IL-10. Scale bar = 200 nm. (D) Size distribution of the isolated exosomes as indicated. (E) qPCR analysis of Il-10 mRNA in the isolated exosomes as indicated. GAPDH served as an internal control. (F) qPCR analysis of Il-10 mRNA in ExoIRES-IL-10 in exosomal RNA degradation assay as indicated. GAPDH served as an internal control. Data are presented as mean ± SEM of three independent experiments. ND, not determined as Ct value greater than 38.

Fig. 1 Construction and features of engineered CAP-Nrf2-Exos. (A, B) Western blot and qRT‒PCR analysis showing the mRNA and protein expression levels of Nrf2 in HEK293T cells. (C, D) Western blot and qRT‒PCR analysis of Nrf2 in the engineered exosomes. (E, F) Western blot and qRT‒PCR analysis of Nrf2 expression in CEP cells after treatment with engineered exosomes. (G) Western blot analysis of Lamp2b, CD9, CD63, TSG101, and β-Actin in HEK293T cells, HEK293T cells transfected with CAP-Nrf2 overexpression plasmids, Exos and CAP-Nrf2-Exos. (H) Exos and CAP-Nrf2-Exos were examined by NTA. (I) TEM analysis of Exos and CAP-Nrf2-Exos isolated from the culture medium of HEK293T cells. Scale bar, 100 nm. (J) Fluorescence images of PKH26-labelled Exos and CAP-Exos internalized by CEP cells. Scale bar, 50 μm and 10 μm. (K) Fluorescence images of DiR-labelled Exos and CAP-Exos in the CEP and IVD in the groups mentioned above. (n = 3, *p 

Figure 2 Exercise-responsive, EV-associated miR-29a-3p in patients with cancer and mouse models (A–C) The morphology of human EVs was observed by TEM, SEM, and IEM. The red arrows point to representative EVs or immuno-gold labeling of CD63. Scale bars: (A and C, left) 500 nm, (A and C, right) 100 nm, and (B, left, middle, and right) 1 μm, 200 nm, and 50 nm. (D and E) Protein immunoblots of EV-non-exercise (NE) or EV-exercise (E), including four typical EV-associated markers. (F) Representative nanoflow cytometry plots of EV markers, including CD9, CD63, and CD81, in both EV-NE and EV-E groups. Quantification of the CD63+ rates in both EV-NE and EV-E groups (n = 6 samples/group). (G) Process flow for EV-associated miRNA sequencing (miRNA-seq) and validation (left). Serum from patients with NSCLC were collected for EV extraction (n = 8 samples/group for miRNA-seq, n = 28 or 20 samples/group for qPCR validation). Heatmap showing differentially expressed miRNAs (right). Top 3 abundant miRNAs are shown. (H) The most abundant miRNAs in the differentially expressed miRNAs and quantification of their prognostic value in NSCLC tumors using TCGA database. (I) Validation of miR-29a-3p expression in serum samples from patients with NSCLC with and without exercise (n = 28 or 20 samples/group). (J) Correlation between estimated erector spinae muscle area and EV-associated miR-29a-3p expression in patients with NSCLC (n = 48 samples/group). (K) Serum miR-29a-3p expression in tumor-bearing mice with and without voluntary exercise (n = 5 mice/group). (L) MiR-29a-3p expression in various freshly isolated cell types in muscle tissues with and without voluntary exercise (n = 3 samples/group). (M) Represented graph of fluorescence distribution of DIR-labeled EVs, non-labeled free EVs, or vehicle in organs and tumors after 24 h of tail vein injection. Positive signals are marked by a spectrum from dark red (weak) to bright yellow (robust). Data are presented as mean ± SD. Significance was calculated with Student’s t test in (F), (H), and (K), univariate Cox analysis in (H), Mann-Whitney test in (I), Spearman test in (J), or one-way ANOVA test in (L). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns, no significance.