Glucose uptake assay

Glucose uptake was measured in fully differentiated C2C12 cells using a non-radioactive fluorometric assay, as previously described (Leira et al., 2002; Zou et al., 2005; Kanwal et al., 2012). Briefly, following differentiation in 6 well plates, cells were serum starved in growth media for 2 h and cells were treated with either vehicle control, IL-15 100 ng/ml, insulin (100 nM), a STAT3 inhibitor (100 μM; S31-201; Sigma), or IL-15 + S31-20 with or without 50 μM 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) (Cayman Chemical) in Krebs-Henseleit buffer containing 0.1% fatty acid free BSA (Sigma) and 2000 mg/L of glucose at 37°C with 5% CO₂(Siddiquee et al., 2007; Kelly et al., 2010). Following 2 h, the cells were lysed in PBS-Triton-X100 buffer and florescence was measured using a microplate reader with excitation 488 nm and emission 520 nm. The concentration of NBDG uptake was calculated from a standard curve of NBDG in lysis buffer. Values were normalized to the protein content present in the cell lysates.