Nampt sirna

A549 human lung carcinoma cells (catalogue no. CCL-185) were purchased from ATCC (Manassas, VA) and grown in DMEM (catalogue no. 11995-065) supplemented with 10% fetal bovine serum (catalogue no. 03-600-511), 100 U/mL penicillin, and 100 U/mL streptomycin (ThermoFisher Scientific, Waltham, MA). Cells were maintained at 37°C in a 5% CO₂ controlled incubator and were passaged every 3 to 4 days at ∼80% confluence. Resazurin sodium salt (ThermoFisher Scientific, Waltham, MA) was re-suspended at 0.16 mg/mL in pH 7.4 PBS and passed through a 0.2 μm syringe filter. Human NAMPT specific carboxyl-terminal enzyme immunoassays (catalogue no. EK-003-80) were purchased from Phoenix Pharmaceuticals (Burlingame, CA). High performance liquid chromatography purified NAMPT siRNA (sense: 5′-CCACCCAACACAAGCAAAGUUUAUU-3′) and scrambled control siRNA (sense: 5′-CCACAACAACAAACGUUGAUCCAUU-3′) were custom synthesized (ThermoFisher Scientific, Waltham, MA). Multiplex human cytokine magnetic bead panels for the detection of IL-1α, IL-1β, IL-1Ra, IL-6, and TNFα (MILLIPLEX MAP, HCYTOMAG-60K) were purchased from EMD Millipore (Darmstadt, Germany).

Using Lipofectamine™ RNAiMAX Reagent (Invitrogen, cat.# 13778-150), hPASMCs were transfected with NAMPT siRNA (L-000458-00), STIM2 siRNA (L-013166-01-0005), ORAI2 siRNA (L-015012-00) or scrambled siRNA (D-001810-02), which were purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO, USA) according to the manufacturer’s protocol.