Fig. 6

Autophagy inhibition activates ubiquitin–proteasome pathway in CuONPs-treated cells. A and C Immunoblotting analysis and quantification of ubiquitinated proteins levels in HUVECs treated with 0, 5, 10, 15 and 20 μg/ml CuONPs for 12 h. GAPDH was used as loading control. B and D Immunoblotting analysis and quantification of ubiquitinated proteins levels in HUVECs treated with CuONPs (20 μg/ml) for 0, 3, 6, 9 and 12 h, respectively. GAPDH was used as loading control. E Representative confocal images of WT and ATG5-KO cells treated with CuONPs (20 μg/ml), respectively. The cells were immunofluorescently stained and analyzed with ubiquitin and SQSTM1 antibody. F and G Immunoblotting analysis and quantification of the levels of NRF2, HMOX1, ubiquitinated proteins, SQSTM1, LC3B and GAPDH (loading control) in WT and ATG5-KO cells treated with 0, 5, 10, 15, 20 and 30 μg/ml CuONPs for 12 h, respectively. H and I Immunoblotting analysis and quantification of NRF2 half-life in CuONPs-treated HUVECs. Cells were treated with tBHQ (10 μM) and CuONPs (20 μg/ml) for 9 h, and then treated with CHX (50 μg/ml) for 0, 1, 2, 3, 6, 9 h, respectively. β-Actin served as loading control. H Immunoblotting analysis and quantification of NRF2 half-life in CuONPs-treated WT or ATG5-KO HUVECs cells. Cells were treated with tBHQ (10 μM) and CuONPs (20 μg/ml) for 9 h, and then treated with CHX (50 μg/ml) for 0, 1, 2, 3, 6, 9 h, respectively. β-Actin served as loading control. In C and D, Student’s t-test was used for statistical significance. In G, I and K, statistical significance was evaluated using one-way ANOVA followed by a Tukey multiple comparison test. All data are representative of three independent experiments. The values are expressed in mean ± S.D. ns not significance; “*”, P ≤ 0.05; “***”, “P ≤ 0.001”