Fig. 2. Insulin induces massive actin rearrangements in BHK-IR cells. (A) Insulin-induced detachment of BHK-IR cells. Cells grown in 24-well plates were serum-starved overnight and stimulated with 100 nM insulin for 4 hours. Detached cells in the supernatant were counted. Bar graphs represent mean ± s.e.m. from a representative experiment with five parallel samples. ^*P<0.05. (B) Insulin-induced changes in the actin cytoskeleton of BHK-IR cells. Serum-starved cells grown on coverslips were stimulated for 30 or 60 minutes with 100 nM insulin, fixed and stained for F-actin with TRITC-phalloidin. (C) y-z and x-y sections of representative noninduced cells or cells induced for 30 minutes with insulin are shown, with their typical F-actin stain. Note the increase in height and the concurrent appearance of actin domes or sheaths in the insulin-treated cells. (D) Serum-starved cells were stimulated for 60 minutes with 100 nM insulin, fixed, stained for F-actin with TRITC-phalloidin and co-stained with anti-annexin-A2 (Anxa2) or anti-phosphotyrosine (pY) antibodies, respectively. Scale bars: 10 µm.