Resveratrol r5010

Anti-PD-L1 (no.13684), anti-GSK-3β (no.12456), anti-N-cadherin (no.4061), anti-SirT1 (no.8469), anti-β-catenin (no.8480) and anti-Snail (no.3895) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Fibronectin (Ab299) was purchased from Abcam (Cambridge, UK). Anti-β-Actin (A1978), anti-Vimentin (V6630) and anti-FLAG (M2) (F1804) were purchased from Sigma (Sigma, Victoria, BC, Canada). Anti-Ac-lysine (sc-32268) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-E-cadherin (13-1700) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-AXIN2 (A2513) was purchased from ABclonal (ABclonal Cambridge, MA, USA). XAV-939 (HY-15147) was purchased from MedChem Express (MedChem Express, NJ, USA). Resveratrol (R5010) and Cycloheximide (239,763-M) was purchased from SIGMA (Sigma, Victoria, BC, Canada). The Flag-SirT1 (#1781), Flag-SirT1 H363Y (#1792), Snail-pGL2 (#31,694), Flag Snail WT (#16,218) and pcDNA-Wnt3A (#35,908) were purchased from Addgene (Cambridge, MA, USA).

As previously reported [18 (link)], H₂O₂ was used to induce acute OS in the airway epithelium. As in our previous study [18 (link)], we have classified donors as being sensitive or resistant to OS based on the concentration of H₂O₂ required to achieve a defined response on epithelial integrity. The donors have different susceptibility to H₂O₂, if cells were exposed to the same concentration of H₂O₂, we would not be able to investigate potential effects of antioxidants on cells from resistant donors, as they have greater ability to resist H₂O₂ exposure. Sensitive donors who are more vulnerable, may benefit from taking antioxidants as a daily supplement to defend against OS.
When the NECs were fully differentiated, cells were treated with 20 μM resveratrol (R5010; Sigma-Aldrich, MI, USA) or astaxanthin (SML0982; Sigma-Aldrich, MI, USA) added to the basal media for 24 hours. NECs were subsequently exposed to 25 mM (sensitive group) or 50 mM (resistant group) H₂O₂ in Hanks’ Balanced Salt solution (HBSS; Sigma-Aldrich, MI, USA) in the apical chamber for one hour to induce an acute OS.