Fig. 1. Expression of G3EGF resulted in cell contact inhibition and reduced cell proliferation. (A) Structures of versican, the versican G3 domain and the G3 domain lacking two EGF-like motifs (G3EGF). (B) Vector- and G3EGF-transfected, and untransfected U87 cells (2x10⁵ cells per well) were cultured on six-well tissue-culture plates to obtain monolayer cultures (–agarose) or on six-well plates coated with 0.6% agarose gel to obtain suspension cultures (+agarose). In the monolayer cultures, the vector-transfected and untransfected cells formed nodules, whereas the G3EGF-transfected cells could only form a monolayer owing to growth contact inhibition. On the agarose-coated plates, the vector-transfected and untransfected cells formed large complexes, whereas the G3EGF-transfected cells formed small conglomerates. (C) Cell growth curves showed that, under monolayer culturing conditions, transfection with G3EGF resulted in a plateau after the cultures reached confluence. (D) On the agarose-coated plates, the vector-transfected cells exhibited higher rate of proliferation than the G3EGF-transfected cells. (E) Expression of G3EGF was confirmed by western blot probed with the monoclonal antibody 4B6. All cell numbers are expressed as the means±SD of three experiments, each performed in triplicate wells,. *P<0.01; **P<0.001.