Fig 1: Accumulative cytotoxic effect of doxorubicin and tumor cells on HUVECs. Representative fluorescent images of actin filaments (phalloidin, green fluorescence) showing morphology of endothelial cells stimulated by different concentrations of doxorubicin as well as MCF7 cells for 24 h at low (A) and high magnifications (B). Representative immunofluorescent images of ICAM1 (red fluorescence) of HUVECs stimulated by different concentrations of doxorubicin as well as MCF7 cells for 24 h at low (C) and high magnifications (D). Representative immunofluorescent images of THP1 (orange fluorescence) and endothelial cell (green fluorescence) adhesion stimulated by different concentrations of doxorubicin in the presence of MCF7 cells at high magnifications (E). Quantitation of cell numbers per view is measured and presented in panel (F). Quantitation of mean fluorescence intensity is measured and presented in panel (G). Corresponding quantitation of THP1 cell adhesion is shown in panel (H). Levels of eNOS (I) and ET-1 (J) in the culture medium were measured. Bars represent 50 and 100 μm. EC: endothelial cell; Dox: doxorubicin. Statistical analysis was performed by one-way ANOVA. n = 5 per group. *P < 0.05, **P < 0.001, ***P < 0.001, and ****P < 0.0001.
Fig 2: Cytotoxic effect of doxorubicin on HUVECs. Representative fluorescent images of actin filaments (phalloidin, green fluorescence) showing morphology of endothelial cells stimulated by different concentrations of doxorubicin for 24 h at low (A) and high magnifications (B). Representative immunofluorescent images of ICAM1 (red fluorescence) of endothelial cells stimulated by different concentrations of doxorubicin for 24 h at low (C) and high magnifications (D). Representative immunofluorescent images of THP1 cell (orange fluorescence) and endothelial cell adhesion (green fluorescence) stimulated by different concentrations of doxorubicin at high magnifications (E). Quantitation of cell numbers per view is measured and presented in panel (F). Quantitation of mean fluorescence intensity is measured and presented in panel (G). Quantitation of THP1 cell adhesion is shown in panel (H). Levels of eNOS (I) and ET-1 (J) in the culture medium were measured. Bars represent 50 and 100 μm. n = 5 per group. EC: endothelial cell; Dox: doxorubicin. Statistical analysis was performed by one-way ANOVA. n = 5 per group. *P < 0.05, ***P < 0.001, and ****P < 0.0001.
Fig 3: NXT-Exo inhibited LPS-induced HMEC-1 dysfunction and apoptosis. (A) The cell viability of HMEC-1 after LPS stimulation were detected by CCK-8 assay. (B) The effects of NXT-Exo on the cell activity of HMEC-1 induced by LPS were detected by CCK-8 assay. (C–E) The expression levels of NO, eNOS and ET-1 in HMEC-1 were measured by ELISA. (F) The apoptosis rates of HMEC-1 were detected by flow cytometry. The horizontal axis B525-A corresponds to the detection of Annexin V staining (Ex = 494 nm, Em = 518 nm), and the vertical axis B610-A corresponds to the detection of PI staining (Ex = 535 nm, Em = 617 nm). (G) The relative protein expression levels of Bax, Bcl-2 and Cleaved caspase 3 were detected by Western blot. Values are expressed as mean ± SD, n = 3. * p < 0.05 and ** p < 0.01.
Fig 4: NXT-Exo inhibited LPS-induced HUVEC dysfunction and apoptosis. (A) The internalization of circulating exosomes from SD rats in HUVEC imaged by confocal microscopy (Scale bar, 10 μm). PKH-26 (Ex: 551 nm, Em: 562∼580 nm) labeled circulating exosomes, Dio (Ex: 484 nm, Em: 496∼520 nm) labeled cell membrane, and Hoechst 33342 (Ex: 346 nm, Em: 455∼470 nm) labeled cell nuclei. (B) The cell viability of HUVEC after LPS stimulation were detected by CCK-8 assay. (C) The effects of NXT-Exo on the cell activity of HUVEC induced by LPS were detected by CCK-8 assay. (D–F) The expression levels of NO, eNOS and ET-1 in HUVEC were measured by ELISA. (G) The apoptosis rates of HUVEC were detected by flow cytometry. The horizontal axis B525-A corresponds to the detection of Annexin V staining (Ex = 494 nm, Em = 518 nm), and the vertical axis B610-A corresponds to the detection of PI staining (Ex = 535 nm, Em = 617 nm). (H) The relative protein expression levels of Bax, Bcl-2 and Cleaved caspase 3 were detected by Western blot. Values are expressed as mean ± SD, n = 3. * p < 0.05, ** p < 0.01.
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