Shp2 is essential for ROCK-dependent rounding in response to RhoA activation. (A) Fluorescent and phase-contrast image of D2 cells transfected with expression vector of pGFP-RhoAV14 and flag-Shp2(C/S) or PTP1B(C/S) (amount of DNA ratio at 1:5) as indicated and plated onto FN-coated coverslip for 30 min. Bar, 20 μm. (B) The percentages of GFP(+) cells spreading on coverslips in total GFP(+) cells were counted and calculated. (C) D2 cells transfected by pEGFP, pMyc-ROCKII(CAT) with or without pflag-Shp2(C/S) (amount of DNA ratio at 1:3:12) were analyzed for spreading on FN-coated coverslips. (D) D2 cells were cotransfected with the expression vectors of GFP and Shp2 E76G or pcDNA3 and were treated with MnCl2 or Y27632, as indicated, before being plated onto FN-coated coverslips. Relative number of GFP(+) cells spreading on FN-coated coverslips within 30 min was determined. (E) Cells transfected by pEGFP, wild-type, or Y722F pMyc-ROCKII with or without pflag-Shp2(C/S) (DNA ratio at1:2:4) were plated onto dish in serum-free medium. After 6 h of incubation, cells were stimulated with or without 20% of serum for 10 min. Cells were imaged by fluorescence microscopy. Bar, 50 μm. (F) The rounding percentage of total GFP(+) cells was calculated. All data are represented as mean ± SD (**, P < 0.05).
