Kc7f2

HPF cells were seeded at a density of 2,000 cells/well in 96-well plates. Then, 24 hrs after seeding, the medium was replaced with fresh medium with and without the following inhibitors at concentrations ranging from 0–100 µM: HIF-1α inhibitor KC7F2 (TOCRIS, Pittsburg, PA), HIF-2α inhibitor TC-S 7009 (TOCRIS, Pittsburg, PA), calcium chelator BAPTA-AM (Life Technologies, Carlsbad, CA), calcium channel blocker verapamil chloride (Sigma, St. Louis, MO), and calcineurin inhibitor cyclosporine A (CsA, R&D Systems, Minneapolis, MN). Cells were treated with these compounds and exposed to normoxic (21% O₂) or hypoxic conditions (1% O₂) for 3 days in the presence of the inhibitors for the entire 3 days. Separate sets of wells were maintained for media and solvent controls. Cell proliferation and cell viability were determined by BrdU assay (EMD Millipore, St Charles, MO) and LDH assay (Thermo Scientific, Waltham, MA), respectively. Cell proliferation and viability graphs were plotted using Origin (OriginLab, Northampton, MA) software with DoseResp function.

Monocytes were primed by culturing 40.000 cells/well in a 96-well plate (Greiner Bio-One^TM) with 2 μg/ml BCG, 2 μM T0901317 (T1317) (Cayman, #71810), 10 μM GW3965 (Cayman, #10054), 5 μM GSK2033 (Cayman, #25443), 500 μM acetyl-CoA (Cayman, # 16160) or 10 ng/ml IL-1β (Peprotech, #200-01A) for 24 h in RPMI supplemented with 10% pooled human AB serum, 5 mM glucose and 1% Penicillin/Streptomycin. In experiments in which inhibitors were used, cells were pre-incubated for at least an hour with 100 nm Torin1 (Cayman, #10997), 20 μM MTA (Cayman, #15593), 10 μM LW6 (Axonmedchem, # 2480), 10 μM KC7F2 (Tocris, #4324), 20 μM Fluvastatin (Sigma, SML0038), 25 μM C75 (Tocris, #2489), 1 μM diacerein (Cayman,# 11710), 200 ng/ml IL-1RA (Peprotech, #200-01RA) prior to priming. The medium was changed after 24 h and cells were rested for 5 days or as indicated. 50% of the medium was refreshed on day 3. On day 6 medium was changed and cells were re-stimulated with either 200 μL RPMI containing 5 μg/ml of Pam3Cys (EMC mirocollection, #L2000), 10 ng/ml of LPS (Sigma) or with medium only. DMSO control was always included when there was a compound dissolved in DMSO. All working solutions were prepared in culture media. The plates were incubated in a 5% CO2 incubator maintained at 37°C. After 24 h supernatants were collected and stored at −20°C until used for cytokine assay.