Fig 1: Evaluation of aortic calcification and mechanism of CMA in the treatment of AS in vivo. (A) In vivo antioxidant performance of CMA. A(i): Representative images of DHE staining (red), Scale bar = 50 μm. Nuclei were stained with DAPI (blue). A(ii): Quantitative analysis of mean DHE fluorescence intensity. A(iii): Vascular cells were isolated, stained with DCFH-DA, and analyzed by flow cytometry for positive rate. A(iv): Serum levels of SOD, GSH-Px, CAT, NADPH, NADP+, and LPO. (B) In vivo anti-inflammatory performance of CMA. B(i): Representative images of iNOs (green) and CD206 (red), Scale bar = 50 μm. Nuclei were stained with DAPI (blue). B(ii): Quantitative analysis of mean iNOs and CD206 fluorescence intensity. B(iii): Quantitative analysis of CD206/iNOs (Mean ratio). B(iv): Serum levels of IL-6, TNF-α, IL-1β, IL-8, and IP10. (C) Serum levels of TC, TG, LDL, and HDL. (D) Serum levels of HMG-CoA, CETP, LCAT, CYP7A1, and HL. (E) Serum levels of leptin, RBP4, MMP-2, ALT, and adipsin. Data are presented as mean ± SEM (n = 6). Significance vs. Control: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001; ns, not significant. Significance vs. Cu-MON: &P < 0.05, &&P < 0.01, &&&P < 0.001, &&&&P < 0.0001; ns, not significant. Significance vs. CMA: #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001; ns, not significant.
Fig 2: Antagonistic effect of Cu-MON on H2O2- or LPS- induced cell senescence. (A) Fluorescence images of RAW264.7 cells following treatment with H2O2 for 1 h and Cu-MON for 24 h. Damaged DNA was stained with γ-H2AX (green), and nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (B) Quantitative analysis of mean γ-H2AX fluorescence intensity. (C) Fluorescence images of RAW264.7 following treatment with H2O2 for 1 h and Cu-MON for 24 h. Senescent cells were stained with SA-β-gal (green), and nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (D) Quantitative analysis of mean SA-β-gal fluorescence intensity. (E) Fluorescence images of RAW264.7 cells treated with H2O2 for 1 h and Cu-MON for 24 h. Mitochondria were stained with JC-1 (green and red), and nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (F) Quantitative analysis of mean JC-1 fluorescence intensity. (G) Cell cycle changes in RAW264.7 cells treated with H2O2 plus different doses of Cu-MON. Relative expression levels of IL-6 (H) and IL-10 (I) in cell culture supernatants of RAW264.7 following 24 h treatment with LPS and Cu-MON. Quantification of the mean fluorescence intensity of Ang I (J) in RAW264.7 cells treated with LPS plus different doses of Cu-MON. Data are presented as mean ± SD (n ≥ 3; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).
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