*2.12. Western Blotting*

Cells were cultured in a 6-well plate with a inoculum density of 4 × 10<sup>5</sup> cells/well and total protein was extracted after the treatments described above. Protein extraction was conducted using a total protein extraction kit according to instructions provided by the manufacturer (Thermo Scientific, Ottawa, ON, Canada). A portion of protein was quantified using a BCA protein assay kit (Thermo Scientific). Then proteins were heat-denatured in an SDS-PAGE loading buffer. Proteins were electrophoresed on polyacrylamide gels and electro-transferred to mini-size nitrocellulose membranes (Bio-Rad, Laboratories Ltd., Montreal, QC, Canada). The immunoreaction was achieved by incubation of the membranes, previously blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% of Tween 20 (TBST), followed by incubation with rabbit anti-Nrf-2 (Thermal Scientific, cat. #PA5-68817), rabbit anti-ZO-1 (Thermal Scientific, cat. #61-7300), rabbit anti-claudin-1(Thermal Scientific, cat. #34-1700), rabbit anti-claudin-3 (Thermal Scientific, cat. #51-9000), and rabbit anti-occludin (Thermal Scientific, cat. #71-1500) proteins after being diluted (1:1000, Abcam Inc., Toronto, ON, Canada) overnight at 4◦C. Afterward, it was washed five times with TBST. Detection of the immune complexes was performed with a horseradish peroxidase-conjugated secondary antibody (1:5000, goa<sup>t</sup> anti-rabbit, Jackson ImmunoResearch Laboratories), then the membrane was washed 5 times, 5 min per time. The ClarityTM Western ECL Substrate was applied to the blot according to the manufacturer's recommendations (Bio-Rad Laboratories Ltd.). The chemiluminescent signals were captured using a ChemiDoc MP imaging system (Bio-Rad Laboratories Ltd.), and Image Lab 6.0 was used to quantify the intensity of the bands (Bio-Rad Laboratories Ltd.). β-actin (from mouse, Thermal Scientific, cat. #AM4302) was set as the internal reference.

Note. ZO-1: zonula occludens-1, SOD: superoxidedismutase, HO-1: hemeoxygenase-1, CAT: catalase, GSH-Px: glutathione peroxidase, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, bp: base pair.

## *2.13. Statistical Analysis*

Data were presented as means ± standard deviations (SD). Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Differences between the means were evaluated by one-way ANOVA. Duncan's multiple range test was used. Level of significance was set at *P* < 0.05.
