Fig. 3. PI3K-signalling controls cortical actin organisation. (A) Actin staining of S2R⁺ cells after treatment with DMSO vehicle (Ctrl), LY294002 (100 µM) and wortmannin (100 nM). Cells were treated for 10 minutes and then fixed and stained with Rhodamine-phalloidin. Arrowheads indicate regions of cell retraction to leave protrusions. Bar, 50 µm. (B) Images of time-lapse movies of S2R⁺ cells treated with DMSO, LY294002 (100 µM) and wortmannin (100 nM). Cells were filmed in phase-contrast on a time-lapse microscope using a 100x oil-immersion lens. Frames were acquired every 10 seconds for 30 minutes with inhibitor treatment after 10 minutes of filming (designated 0' in the figure). Snapshots are shown at 0, 5 and 10 minutes. The corresponding kymographs show cells from 3 minutes before until 10 minutes after insulin stimulation and were constructed from pixel intensities taken along the lines depicted in the images on the left. Bar, 50 µm. (C) Immunoblotting of P-Akt and total Akt in untreated S2R⁺ cells and cells treated with insulin (for 10 minutes) with and without pre-treatment (for 10 minutes) with LY294002 (100 µM) or wortmannin (100 nM). (D) Immunoblotting of P-Akt, -actin and P-cofilin in S2R⁺ cells treated for 10 minutes with LY294002 (100 µM) and insulin (10 µg/ml), alone and in combination. Relative abundance is represented as a percentage of the control below the blot and is the average calculated from densitometry measurements of blots from three independent experiments.