Drugs were added to MCF7-EGFP-α-tubulin cells in 1% FBS for 4 h after which coverslips were placed in recording media (10% FBS-DMEM, lacking phenol red and sodium bicarbonate, but supplemented with 15 mM HEPES, 3.5 g/L glucose, Oxyrase (1:50 dilution), and DL-lactate (10 mmol/L)). Cells were visualized using a 100× Nikon Plan Apo objective (N.A. 1.4, oil immersion) at 37°C on a Nikon Eclipse E800 microscope (Nikon; Tokyo, Japan) equipped with a CoolSNAP HQ2 camera (Roper Scientific GmbH, Ottobrunn, Germany). Images were taken at 4-s intervals for 2.5 min using an exposure time of 600 ms, no binning, and an 8-bit image auto scale using Metamorph software (Molecular Devices, Sunnyvale, CA) [28 (link)]. Plus ends of microtubules were tracked using Igor Pro 6.22A: Microtubule Life History Analysis Package designed by Dr. Emin Oroudjev (University of California Santa Barbara, 2010). Dynamic instability parameters were determined as described [28 (link)]. A minimum of 30 microtubules were measured from three independent experiments per condition, and reported as mean ± SEM.
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