The effect of insulin on GP decrease is augmented by phospholipase C inhibitor, U73122. Cells stably expressing WT 2031-ACP-IR cultured on glass bottom dishes for 2 d were labeled with CoA-PEG5-NR. The excess dye was washed, 1 ml of 37°C serum-free medium containing 15 mM Hepes was added to the dish, and then the dish was placed on the stage of a Zeiss LSM 780 confocal microscope warmed at 37°C 5 min before the observation. 1 min before observation, 1 μl of DMSO (control) or 1 μl of 1 mM U73122 (final concentration, 1 μM) was added to the dish. Spectrum images in the range of 561–695 nm were captured with 15 channels (step 8.9 nm, 4 times line-average) using lambda mode excited at 514 nm with 1.5% of Argon laser power every 10 s for 7 min, and 100 μl of serum-free medium (control) or insulin in serum-free medium (final concentration, 100 nM) was added to the dish at 120 s. The differential delta GP with insulin treatment of both control and U732122 over the basal control-treated delta GP of each sample was plotted. The basal control-treated delta GP was estimated as the mean delta GP of 8 time-lapse data sets for both control and U732122. Data represent means ± SEM of eight time-lapse data sets for each.