Fig 1: CuS/EPDA tumor targeting mechanism. (A) Cell viability assay of lung metastatic H-1993 cells after treatment with CuS (2mg/mL) or CuS/EPDA (2–4mg/mL) for 4h (Scale bar, 20 μm). (B and C) cytotoxicity evaluation of CuS/EPDA NCs in H-1993 and A549 cells under neutral or hypoxic pH. Cells were treated with CuS/EPDA NCs (0.5–10μg/mL−1) for 4h. Data represent the mean±SD of three independent experiments (*P < 0.05; **P < 0.01). (D–F) Flow cytometry analysis of intracellular cytokines TNF-A, IL-6 and IL-12 levels in NC-treated mice. Serum from mice was isolated 48–72h following treatment. Data represent the mean ±SD of three independent experiments. (G and H) DC maturation analysis using flow cytometry after staining with CD11c, CD80 and CD86. Data represent the mean ±SD of three independent experiments (**P < 0.01). (I and J) Immunofluorescence staining of immunosuppression biomarkers iNOS and Arg1 in tumor tissues after CuS and CuS/EPDA incubation. Confocal microscopy images of Arg1 (green). iNOS labeled cells (red) and DAPI (blue). Images were captured using Carl Zeiss fluorescence confocal microscope. Data represent the mean±SD of three independent experiments (*P < 0.01). (K) Intracellular ROS assay (Deepred, ab186029, Abcam) of lung metastatic H-1993 cells after treatment with CuS (2mg/mL) or CuS/EPDA (2–4mg/mL) for 4 h (scale bar, 20 μm). Differences were considered statistically significant at p < 0.05. Statistically significant data are indicated by asterisks.
Fig 2: CuS/EPDA tumor targeting mechanism. (A) Cell viability assay of lung metastatic H-1993 cells after treatment with CuS (2mg/mL) or CuS/EPDA (2–4mg/mL) for 4h (Scale bar, 20 μm). (B and C) cytotoxicity evaluation of CuS/EPDA NCs in H-1993 and A549 cells under neutral or hypoxic pH. Cells were treated with CuS/EPDA NCs (0.5–10μg/mL−1) for 4h. Data represent the mean±SD of three independent experiments (*P < 0.05; **P < 0.01). (D–F) Flow cytometry analysis of intracellular cytokines TNF-A, IL-6 and IL-12 levels in NC-treated mice. Serum from mice was isolated 48–72h following treatment. Data represent the mean ±SD of three independent experiments. (G and H) DC maturation analysis using flow cytometry after staining with CD11c, CD80 and CD86. Data represent the mean ±SD of three independent experiments (**P < 0.01). (I and J) Immunofluorescence staining of immunosuppression biomarkers iNOS and Arg1 in tumor tissues after CuS and CuS/EPDA incubation. Confocal microscopy images of Arg1 (green). iNOS labeled cells (red) and DAPI (blue). Images were captured using Carl Zeiss fluorescence confocal microscope. Data represent the mean±SD of three independent experiments (*P < 0.01). (K) Intracellular ROS assay (Deepred, ab186029, Abcam) of lung metastatic H-1993 cells after treatment with CuS (2mg/mL) or CuS/EPDA (2–4mg/mL) for 4 h (scale bar, 20 μm). Differences were considered statistically significant at p < 0.05. Statistically significant data are indicated by asterisks.
Fig 3: YK-4–250 retains the antioxidant properties of tempol and demonstrates increase ACE2 expression. (A) Intracellular ROS levels in Caco-2 cells were measured following treatment with YK-4–250, telmisartan, or tempol (50 µM, 2 h). Data are presented as Mean ± SEM of N = 3 independent biological experiments. Cells were stimulated with tert-butyl hydroperoxide (100 µM, 30 min) to induce oxidative stress, and ROS generation was quantified using the Abcam Cellular ROS Assay Kit (Deep Red, ab186029) with fluorescence detection at 660–720 nm. (B) A549 cells were cultured for 24 h before treatments then continued culturing for an additional 24 h before total RNA isolation. ACE2 qRT-PCR was performed and compared to vehicle-only control.
Supplier Page from Abcam for Cellular ROS Assay Kit (Deep Red)