Fig. 3
TiO2-NP activated ADAM17 directly, contributing to a rapid claudin-5 protein degradation. a ADAM17 kinetics assay was performed by fluorometric method in accordance with manufacture’s protocol. The releasing fluorescence from FRET substrate is relative to ADAM17 activity. The basal ADAM17 activity of HREC lysate (vehicle control) was showed in brown curve. In combination with TiO2-NP (500 ng/mL), an increasing fluorescence illustrated the induction of ADAM17 activity by TiO2-NP (orange curve), whereas the fluorescence signals were prevented by GM6001 (dark green curve) and TAPI-2 (light green curve). b HREC lysate was prepared and aliquoted (20 μg protein/vial), then incubated with TiO2-NPs (50–1000 ng/mL) at 37 °C for 3 h. Another vial, without TiO2-NP treatment, was used as control. Next, the reaction was stopped by the addition of SDS-PAGE sample loading buffer. The amount of claudin-5 was detected by immunoblotting. Data showed an apparent protein degradation of claudin-5, whereas ADAM17 protein level was not changed, whatever the presence or absence of TiO2-NP treatment. c TiO2-NP-mediated claudin-5 degradation could be prevented by GM6001 and TAPI-2. These data provide substantial evidence to support the TiO2-NP-mediated claudin-5 degradation, involving the activation of ADAM17. (*p < 0.05, **p < 0.01, ***p < 0.001, indicates statistically significant difference from the control treatment; ## p < 0.01 indicates statistically significant difference from the TiO22-NP-treated group)