Measurement of lactic acid production, glucose uptake and cellular ATP levels

A549 and H446 cells (1×104 cells/well) were seeded into 6-well plates and cultured in DMEM. A549 and H446 cells were treated with shikonin (0, 10, 20 or 50 µM) for 24 h at 37°C or transfected A549 cells were treated with 50 µM shikonin for 24 h at 37°C, then cell culture medium was subsequently collected and centrifuged at 8,000 × g for 5 min at room temperature. The supernatant was used to measure lactate or glucose concentrations, or cellular ATP levels. Extracellular lactate levels were measured using a lactate analysis kit (cat. no. E4341; BioVision, Inc.) according to the manufacturer's protocol. Cell lysates were used to measure glucose levels using a glucose assay kit (cat. no. K686; BioVision, Inc.) according to the manufacturer's protocol. ATP levels were measured using the CellTiter-Glo® luminescent cell activity assay kit (cat. no. G7572; Promega Corporation) according to the manufacturer's protocol. The relative value of lactate, glucose uptake and ATP levels were normalized to the control group (set to 1).

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