Poly da dt naked

For oxidative stress, hydrogen peroxide (H₂O₂) solution (30%) (Fujifilm Wako, 084‐07441) was diluted 1000‐fold with the culture medium and added to both the apex and bottom of the ALI. Cells were preincubated for 1 h with Mito‐TEMPO (Hydorate, Cayman Chemical 16621) or 2 h with the STING inhibitor, H‐151 (InvivoGen inh‐h151) and then stimulated with H₂O₂.
For intracellular DNA provocation, NCI‐H292 cells were transfected with 0.5 μg of poly(dA:dT) naked (Invivogen, tlrl‐patn) using the FuGENE HD Transfection Reagent (Promega, E2311) and incubated for 24 h. 2′3′ cGAMP (InvivoGen, tlrl‐cga23‐s) was dissolved in double‐distilled water at 1 mg/mL and stored at −20°C until use. Cells were stimulated with 2′3′ cGAMP for 16 h. MitoTEMPO (Cayman Chemical, 16621) was preadministered to cells 1 h before H₂O₂ stimulation. Thapsigargin (Cayman Chemical, 10522) was dissolved in dimethyl sulfoxide at 1 mM and stored at −20°C until use. Thapsigargin, which can trigger mitochondrial DNA release into the cytosol,¹³ was diluted with culture medium to appropriate concentrations, incubated for 4 h, and further cultured in serum‐free RPMI‐1640 for 20 h. Cells were incubated with a medium containing recombinant human transforming growth factor‐alpha (TGF‐α; R&D 239‐A‐100), an EGF receptor (EGFR) agonist for 6 h.