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Fig. 4. The Rho/ROCK pathway is involved in mediating insulin-induced morphological changes in BHK-IR cells. (A) The active RhoA mutant RhoQ63L induces an insulin-like phenotype. BHK-IR cells were transfected with a Myc-tagged RhoQ63L construct and co-stained with anti-Myc-antibodies and TRITC-phalloidin or anti-annexin-A2 antibodies. Note the appearance of the typical `insulin phenotype', i.e. cell rounding and the formation of actin domes upon RhoQ63L expression. Scale bars: 10 µm. (B) Rho/ROCK activity is required for insulin-induced morphological changes. Serum-starved cells grown on coverslips were left untreated or preincubated either with the cell-permeable Rho-inhibiting C3 exotoxin or with two different, structurally unrelated ROCK inhibitors, Y27632 and H-1152, prior to insulin stimulation for 30 minutes. The fixed cells were stained for F-actin and morphological changes in the actin cytoskeleton were quantified. **P<0.0005.
