Fig. 2. The kinetics of actin remodelling and cofilin phosphorylation following an acute stimulus. (A) S2R⁺ cells stained for F-actin (Rhodamine-phalloidin) and nuclei (DAPI) are shown at various times after insulin stimulation (10 µg/ml). Bar, 50 µm. (B) Images from a time-lapse movie of S2R⁺ cells stimulated with insulin (10 µg/ml) for the times indicated. Cells were filmed in phase-contrast on a time-lapse microscope using a 100x oil-immersion lens; frames were acquired every 20 seconds, 10 minutes prior to insulin addition and for 30 minutes after addition. Images are representative of five independent experiments. Bar, 50 µm. Right panel shows a kymograph of the movie from which the presented images were taken, generated from pixel intensities along a line transecting the cell membrane (shown in first image). It shows increased protrusion dynamics after insulin stimulation. (C) F-actin immunostaining (Rhodamine-phalloidin) of serum-starved Kc167 cells stimulated with insulin (10 µg/ml) for the indicated times. Bar, 50 µm. (D) Cofilin is transiently phosphorylated in S2R⁺ cells in response to insulin. S2R⁺ cells were grown in serum-free medium overnight and stimulated with insulin at 10 µg/ml for the indicated times. Lysates were prepared and immunoblotted with antibody against P-cofilin. Blots were analysed by densitometry. Values represent the mean P-cofilin signal from two experiments after normalising with respect to -actin levels. Error bars represent the standard deviation. (E) Time course of Akt activation in insulin stimulated S2R⁺ cells. S2R⁺ cells were maintained in Schneider's serum-free medium overnight, and then stimulated with bovine insulin (10 µg/ml) for the times indicated. Immunoblotting was used to assess levels of P-Akt and total Akt.