Figure 1.

Expression of RHOA and ROCK2 in human transformed and GC cell lines.

A, Human GC cell lines (AGS, MKN45) and HEK293 cells transformed with large T-antigen (TAg) express total RHOA but not active RHOA protein. Cells were grown to confluency followed by GST-pulldown from TCLs using recombinant rhotekin-GDS protein as bait for precipitation of active GTP-bound RHOA protein. Precipitated active RHOA was then detected together with total RHOA protein (“input” = TCL before pulldown) by Western blots (WB) using a RHOA p21 Ab. O.D. values from bands in gels were normalized to HSP90 (loading control) and calculated as -fold ± S.E. compared with control (n = 3 per cell line; input P= .039 and pulldown P= .021 MKN45 vs. AGS, Kruskal Wallis test). Quantitative analyses and representative gels are shown.

B, Cells were treated with vehicle (PBS, −) or fasudil (FASU, +) for 30 hours before WB of total cell lysates. O.D. values from bands in gels were normalized to HSP90 (loading control) and calculated as -fold ± S.E. (n = 3 per cell line; P= .0006 FASU vs. PBS, t-test) compared with vehicle control. Quantitative analyses and representative gels are shown.

C, ROCK1/2 inhibition promotes cell death. Cells were treated with vehicle (PBS) or increasing concentrations of fasudil for 48 hours, and cell viability was measured by colorimetric MTT assay after 2 days. O.D. values were calculated as % ± S.E. (n = 3 per cell line, P= .0078 and P= .0001 FASU vs. PBS, Mann-Whitney U test) compared with vehicle control.