Figure 2

MSOR mitigate different parameters associated with cellular transformation. (A) The influence of mono- and trivalent wild-type RBDs on Ras-driven invasion was analyzed after transient transfection of COS-7 cells with expression constructs for K-RasG12V and E1, E1-R1 or E1-R3 and subsequent transmigration of transfected cells through a Matrigel® layer. The figure shows the average of three independent experiments. (B) The impact of mono- and trivalent wild-type RBD probes on c-Met-stimulated anchorage-independent growth was investigated by seeding NIH3T3-TM cells transiently expressing E1, E1-R1 or E1-R3 into soft agar and subsequent culture in the presence or absence of 25 ng/ml NGF. Colony formation was evaluated by counting of colonies in at least ten arbitrarily selected vision fields. The figure shows the average of three independent experiments. (C) Effect of the trivalent wild-type RBD construct on the Ki-RasG12V-induced protease gene expression. COS-7 cells were transiently transfected with a plasmid encoding E1 alone or an expression construct for K-RasG12V along with E1 or E1-R3. The impact of RBD constructs on K-RasG12V-stimulated expression of different proteases was analyzed on a custome oligonucleotide microarray. Signals were assessed densitometrically and normalized to the E1 expression level. See Material and methods for a more detailed description. Data are derived from three independent experiments. (D) Consequences of mono- and trivalent wild-type RBD probes on the K-RasG12V-stimulated induction of the human MMP1-promoter. NIH3T3 cells were transiently transfected with E1, E1-R1 or E1-R3 together with an expression construct encoding K-RasG12V as indicated. Then, a MMP-1-firefly-luciferase reporter plasmid was co-transfected along with a reference renilla luciferase construct and the relative luciferase activity was determined. The figure shows the average of three independent experiments each performed in duplicates.