Figure 1
Characterization of nanosized copper particles (nano Cu). A and B, Transmission electron microscope images of nano Cu. C and D, Scanning electron microscopy images of nano Cu. Scale bars: A, 200 nm; B, 50 nm; C, 2.00 μm; D, 500 nm. E, Distribution of nano Cu average size in culture medium was determined by dynamic light scattering image.
Figure 2
Effect of nanosized copper particles (nano Cu) on cell viability in mesangial cells (MCs). A culture medium (0, 1, 10, 30, and 50 μg/mL nano Cu) was used to treat the MCs for 1, 2, 3, 6, and 12 h. A CCK-8 assay was used to measure the viability of the cells, and the outcomes are reported as the absorbance percentage relative to the control group. Data are reported as means±SD (n=6/group). **P<0.01 vs control group (ANOVA).
Figure 3
Effect of nanosized copper particles (nano Cu) on cell apoptosis in mesangial cells (MCs) by flow cytometry analysis. Cells were exposed to 0, 1, 10, 30, and 50 μg/mL nano Cu for 3 h. A, Control group. B-E, Cells treated with 1, 10, 30, and 50 µg/mL nano Cu, respectively. F, Bar graph of the results. Data are reported as means±SD, from three independent experiments. **P<0.01 vs control group (ANOVA).
Figure 4
Effect of nanosized copper particles (nano Cu) on cell morphological characteristics of apoptosis by Hoechst 33342 staining in mesangial cells (MCs). Cells treated using nano Cu for 3 h were stained with Hoechst 33342 and images were observed under the fluorescence microscope. A, Control group. B-E, Cells treated with 1, 10, 30, and 50 µg/mL nano Cu, respectively. The white arrows show nuclear condensation and fragmentation in MCs. Scale bar, 50 µm. F, Bar graph of the results. Data are reported as means±SD, from three independent experiments. **P<0.01 vs control group (ANOVA).
Figure 5
Effect of nanosized copper particles (nano Cu) on the activity of caspase-3 in mesangial cells (MCs) by ELISA. MCs were treated with different concentrations of nano Cu (0, 1, 10, 30, and 50 μg/mL) for 3 h. Data are reported as means±SD, from three independent experiments. **P<0.01 vs control group (ANOVA).
Figure 6
Effect of nanosized copper particles (nano Cu) on the generation of reactive oxygen species in mesangial cells (MCs) by DCFH-DA staining with flow cytometry analysis. Cells were exposed to 0, 1, 10, 30, and 50 μg/mL nano Cu for 3 h and fluorescence intensity was measured. A, Control group. B-E, Cells treated with 1, 10, 30, and 50 µg/mL nano Cu, respectively. F, Bar graph of the results. Data are reported as means±SD, from three independent experiments. *P<0.05, **P<0.01 vs control group (ANOVA).
Figure 7
Effect of nanosized copper particles (nano Cu) on autophagy in mesangial cells (MCs). A, Effect of nano Cu on the formation of acidic vesicular organelles in MCs. Cells were treated with nano Cu for 3 h, stained by acridine orange. The formation of acidic vesicular organelles was examined under fluorescence microscopy. Scale bar is 50 µm. B, Cells were subjected to Western blotting analysis with anti-Beclin-1, anti-LC3, and anti-p62 antibodies, after exposing to 0, 10, 30, and 50 μg/mL nano Cu for 3 h. GAPDH served as the loading control. C, The corresponding linear diagram of immunoblotting quantitation was shown. Each reported value represents as means±SD. From three independent experiments. D, Effect of 3-MA on the cytoxicity of nano Cu in MCs. A CCK-8 assay was used to detect the effect of 3-MA on the cytoxicity of nano Cu on cell viability in MCs. Cells were exposed to cell culture medium, 1, 10, 30, and 50 μg/mL nano Cu, and 30 μg/mL nano Cu+3-MA for 3 h. Data are reported as means±SD (n=6/group). *P<0.05, **P<0.01 vs control group. ##P<0.01 vs 30μg/mL nano Cu group.
Figure 8
Effect of nanosized copper particles (nano Cu) on the Akt/mTORp70S6K pathway in mesangial cells (MCs). A, MCs were exposed to 0, 10, 30, and 50 μg/mL nano Cu for 3 h and subjected to western blotting analysis with anti-Akt, anti-p-Akt, anti-mTOR, anti-p-mTOR, anti-p70S6K, and anti-p-p70S6K antibodies. GAPDH served as the loading control. B, Corresponding linear diagrams of immunoblotting quantitation. Data are reported as means±SD, from three independent experiments. *P<0.05, **P<0.01 vs control group (ANOVA).
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