Isoproterenol

Immortalized gingiva keratinocyte (KC-TERT, OKG4/bmi1/TERT, Rheinwald laboratory, Boston, USA) (passage 10 to 20) and fibroblast (Fib-TERT, T0026, ABM, Richmond, BC, Canada) (passage 10 to 20) cell lines were used for constructing RHG. The RHG model, consisting of a fibroblast-populated collagen hydrogel (collagen I derived from rat tail) overlaid with keratinocytes (0.5 x 10⁶ cells) was cultured exactly as previously described (Kosten et al., 2015 (link)). In brief, RHG were cultured in six-well transwell inserts (pore size 0.4μm, 24mm, Corning, USA), first submerged in culture medium for 3 days and then lifted to the air-liquid interface for an additional 10 days to induce epithelial differentiation and stratification in culture medium containing DMEM/Ham’s F12 (3/1) (Gibco, Grand Island, USA) supplemented with 1% Fetal Clone III (HyClone, GE Healthcare, Chicago, USA), 1% PS (Gibco, Grand Island, USA), 0.1µM insulin, 1µM hydrocortisone, 1µM isoproterenol, 10µM L-carnitine, 10mM L-serine, 0.4mM ascorbic acid, and 2ng/mL epidermal growth factor. All agents were purchased at Sigma-Aldrich (St. Louis, USA) when not specified. One day prior to bacteria exposure, PS and hydrocortisone were omitted in RHG culture medium for some experimental conditions as indicated below.

Isoproterenol (ISO; Sigma Aldrich, I5627), milrinone (MIL; Tocris, 1,504), omecamtiv mecarbil (OME; Adooq Biosciences, A11206), verapamil (VER; Tocris, 0654), and Propranolol (PRO; Sigma Aldrich, P0884) solutions were prepared freshly before use. Isoproterenol and verapamil were solubilized in distilled water (Gibco, 15230-071) as 10 mM stock. Propranolol and omecamtiv mecarbil were solubilized in DMSO (Sigma, D2650) as 10 mM stock and milrinone as 50 mM stock. They were diluted in L-15 medium (Gibco, 11415-049). During measurements, the test compounds were administered directly to the wells, diluting them to the final concentrations. The amount of DMSO in the final concentrations of omecamtiv mecarbil and Propranolol did not exceed 0.003%. For milrinone, the amount of DMSO in the final concentrations was 0.002, 0.02, and 0.2%.

Colchicine (10 µM in DMSO, Sigma-Aldrich), Phenylephrine (100 µM in culture media, Sigma-Aldrich), Isoproterenol (1 µM in DMSO), Nocodazole (10 µM in DMSO, Thermo Fisher), Puromycin (10 µg/mL solution, A.G. Scientific), Cycloheximide (20 µg/mL in DMSO, Sigma-Aldrich), Blebbistatin (5 µM in DMSO, Cayman Chemical), Latrunculin A (10 µM in DMSO, Abcam), Y27632 (10 µM in DMSO, Sigma-Aldrich). Cells were treated with Colchicine, Nocodazole, or DMSO for 3 h prior to treatment with PE.