Ezatiostat

In order to demonstrate the secretion of GSTpi by the bronchial epithelial cells, apical media of ALI-cultured Calu-3 cells (ATCC No. HTB-55, Lot. 61449062) were assayed. Briefly, cells at passage 23–30 were cultured (3 × 10⁶ cells/cm²) in DMEM nutrient mixture F-12 (Thermo Fisher Scientific), containing 2 mM l-glutamine (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin (Lonza) and 10% foetal bovine serum (Hyclone, GE Healthcare, IL, USA) at 37 °C and 95% humidified atmosphere of 5% CO₂ in air, onto 6.5 mm-Transwell^® insert (pore size 0,4 μm, Corning, NY, USA). After 7 days on ALI, cells were apically covered with 100 μL PBS (Lonza, Basel, Switzerland) during 24 h and apical media were collected and keep on ice until use.
Calu-3-derived apical medium (25 μL) was used as a potential source of GSTpi in the proteolysis assays mentioned above. Some samples were preincubated with 0.5 mM of the specific inhibitor of GSTpi TLK199 (beyond this concentration the drug is not soluble, Ezatiostat, Sigma). In addition, the presence of GSTpi in the apical medium was detected by Western blot, using the rabbit polyclonal antibody anti-human GSTpi (Dilution 1:2500, anti-GST3, Abcam, Cambridge, UK).