5 aminoimidazole 4 carboxamide ribonucleotide aicar

In vitro cell experiments were carried out in Primaria plates (BD Falcon) for uroepithelial cells, TERT-NHUC, and polystyrene (Costar) for 5637 and THP1 differentiated cells. Nearly confluent cells, 70–80%, were treated with freshly prepared metformin (4 mM, Sigma-Aldrich) for 24 h, deionized water served as vehicle control. 4 mM metformin concentration was found to be optimal for both the cell lines based on the dose response expression profiles of CAMP and RNASE7 expression (Fig. S1).
5' adenosine monophosphate-activated protein kinase (AMPK) activator 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, Sigma-Aldrich) or AMPK inhibitor, Compound C (20 µM, Sigma-Aldrich) were used and cells were treated for 24 h. In blocking experiments, Compound C was added 1 h prior to adding AICAR or metformin. Chinese hamster ovary cell line expressed growth/differentiation factor 15 peptide (GDF15, 50 ng/ml, R&D systems) was treated to the cells for 24 h. For TRPA1 RT-PCR or imaging experiments, cells were stimulated with the activator, allyl isothiocyanate (AITC, 10 µM, Sigma-Aldrich) and inhibitor A-967079 (A96, 20 µM, Sigma-Aldrich), for 24 h for mRNA analysis. Equal amount of DMSO served as vehicle in control cells.

Human bronchial epithelial cells BEAS-2B cells (Sigma) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml) in 5% CO₂ and humid air at 37^°C. Human small airway epithelial cells SAECs (Lonza) were maintained in Small Airway Epithelial Cell Growth Medium (SAGM; basal medium plus growth supplements, Lonza). Cells were used between passage 4 and 10. Cells were exposed to CSE (0.25% for SAECs and 0.5% for BEAS-2B based on their viability) for 24 hours in the presence or absence of 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, Sigma) and Compound C (5 μM, Sigma).

The human renal proximal tubular epithelial cell line HKC8 was obtained from Dr. L. Rausen (Johns Hopkins University, Baltimore, MD) and was maintained in Dulbecco's Modified Eagle Medium supplemented with Ham's F12 medium (DMEM/F12; Invitrogen). DMEM/F12 was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (WelGENE, Daegu, Republic of Korea). Before experiments, cells were maintained in medium containing 5–30 mM D-glucose. To increase PGC1α activity, 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; Sigma-Aldrich St. Louis, MO, USA) or 1 mM metformin (Sigma-Aldrich, St. Louis, MO, USA) was added to the culture media.

H9c2 cells (Korean Cell Line Bank, Seoul, South Korea) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 50 U/ml penicillin, and 50 g/ml streptomycin (Invitrogen). Cells were propagated at 37°C with 5% CO₂ and subcultured in 24-well plates until reaching 90% confluency, after which they were transfected with rat sesn2-targeting siRNA or scrambled siRNA (Genolution, Seoul, South Korea) using Lipofectamine™ according to the user manual (Invitrogen). After 1 day, the efficiency of sesn2 knockdown was determined by Western blotting. LPS (Sigma-Aldrich, MO, USA), 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (Sigma-Aldrich), and N-acetyl-cysteine (NAC) (Sigma-Aldrich) were dissolved in phosphate-buffered saline (PBS) (Biosesang, Seoul, Korea) and used to treat the transfected H9c2 cells. All additives were cotreated; cells were not serum starved.