Fig. 4. The effect of MK-TRAP on anchorage-independent growth of G401 and CMT-93 cells. (A) M1 and C1 cells (3000 cells per dish) were plated in 35-mm dishes in soft agar. Two weeks later, representative colonies were photographed. (B) The total numbers of colonies of M1 cells versus C1 cells were counted and calculated as the mean of three independent experiments (n=3); error bars indicate ± s.e.m.; ****P<0.001. (C) Exogenous MK (10 ng/ml or 100 ng/ml) was added to both upper and bottom gels to rescue decreased anchorage-independent growth of M1 cells. The results are presented as means (n=3); error bars, ± s.e.m.; **P<0.01. (D) G401 cells transiently transfected with MK-TRAP or empty vector (control) were subjected to soft agar colony-formation assay, and the numbers of colonies with a diameter () >50 µm or >200 µm in each group were counted and calculated as means. n=3; error bars, ± s.e.m.; *P<0.05; ****P<0.001. (E) MK-TRAP-containing medium was produced by transient overexpression in COS7 cells and then added to both upper and bottom gels, in order to evaluate effects on anchorage-independent growth of G401 cells and CMT-93 cells. Left, G401 cell line; right, CMT-93 cell line. n=3; error bars, ± s.e.m.; ***P<0.005; ****P<0.001. (F) Goat anti-MK antibody (15 µg/ml) was added to upper gels to evaluate its effects on anchorage-independent growth of G401 cells and CMT-93 cells. Left, G401 cell line; right, CMT-93 cell line. n=5; error bars, ± s.e.m.; ****P<0.001. (G) CMT-93 bulk stable transformants were generated as described in the Materials and Methods section. Cells were inoculated subcutaneously into nude mice. The volume of the tumors was measured at the indicated intervals (left panel). The data are shown as the means ± s.e.m. (n=8). Twenty-five days later, the tumors were isolated and weighed immediately (right panel). Data are given as the means ± s.e.m. (n=8); *P<0.05.