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. 2012 Aug 20;7(8):e43662. doi: 10.1371/journal.pone.0043662

Figure 2. Time courses of HA-GLUT4-eGFP intensity and microtubule density increase.

Figure 2

(A) Insulin increases microtubule density in the region of the cell illuminated by TIRF microscopy. Adipocytes transfected with constructs either to visualize the GLUT4 or microtubule time course were serum-starved prior to stimulation with 100 nM insulin at t = 0 min. Images were acquired using TIRF microscopy at 1 frame per 10 s. Intensity or density increase for each cell was normalized from 0 (average intensity or density prior to insulin addition) to 1 (intensity at last 1 minute or density at last 8 minutes of time course). Plotted is the mean ±95% confidence interval for each point (HA-GLUT4-eGFP, n = 13 cells; mCherry-tubulin, n = 18 cells). (B) Nocodazole pre-treatment decreases the fold change in GLUT4 intensity in response to insulin. Adipocytes expressing HA-GLUT4-eGFP were serum-starved and pre-treated with 0 µM (dark blue, n = 13 cells) or 10 µM (light blue, n = 7 cells) nocodazole for a minimum of 20 min prior to stimulation with 100 nM insulin at t = 0 min. Images were acquired using TIRF microscopy at 1 frame per 10 s. Plotted is the time course of the mean fold intensity increase ±95% confidence interval. There is a significant difference (two-tailed t-test; p-value <0.05) between the two time courses at the half-time for HA-GLUT4-eGFP intensity increase (t = 6.3 min, 0 µM Noc). Due to heterogeneity in the magnitude of the insulin response (Figure 1B inset), a two-tailed t-test performed at t = 25 min does not yield a significant p-value (p-value >0.05). However, since there is a temporal relationship between time points, a new p-value, P, can be calculated by taking this relationship into consideration (see Methods). Comparing the last several minutes of the plateaus gives a significant difference (P<0.05) between the 0 µM Noc and 10 µM Noc time courses. (Inset) Time course replotted to show the intensity increase normalized from 0 (average intensity prior to insulin addition) to 1 (intensity at last minute of time course). Plotted is the mean ±95% confidence interval. Half-times are plotted (0 µM Noc, t 1/2 = 6.3 min, solid black line; 10 µM Noc, t 1/2 = 9.8 min, dashed black line).