The mTOR/GCLc/GSH Pathway Mediates the Dose-Dependent Bidirectional Regulation of ROS Induced by TiO₂ NPs in Neurogenic Cells

Figure 3

(a) ROS levels were measured by FCM after cells were treated with 100 μg/ml TiO₂ NPs and 100 μg/ml TiO₂ NPs+GSH for 24 h. (b–d) Key enzyme activities in the ROS regulation pathways (including SOD, CAT, and GSH-Px) were measured after cells were treated with TiO₂ NPs. Total protein was used as an internal control. (e) Mitochondrial membrane potential was determined by JC-1. Aggregated and monomeric JC-1 concentrations were measured by average fluorescence intensity at 590 nm and 530 nm, respectively. The ratio of 590/530 nm indicated the changes of mitochondrial membrane potential. (f) ATP levels were measured after cells were treated with TiO₂ NPs for 24 h. (g, h) Intracellular superoxide anion and mTOR protein expression levels were measured after cells were treated with 100 μg/ml of TiO₂ NPs and 100 μg/ml of TiO₂ NPs+50 μM of Mn(III)TBAP for 24 h. (i) Intracellular superoxide anion levels were measured after cells were treated with 1 μg/ml of TiO₂ NPs and 1 μg/ml of TiO₂ NPs+RAPA for 24 h. (j, k) Mitochondrial membrane potential and mTOR protein expression levels were measured after cells were treated with 1 μg/ml of TiO₂ NPs for 3 h. (l, m) SOD activities and superoxide anion concentrations were detected after cells were treated with 1 μg/ml of TiO₂ NPs for 3 h. Values are presented as , and each assay was repeated three times. , , .

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