Figure 5

A non-toxic MSOR-variant efficiently blocks Ras-induced signaling and transformation. (A) Confocal images of COS-7 cells transiently expressing the mono- and trivalent low affinity RBD-R59A/N64D probes (left panel). Scale bar 10 μm. Cell death analysis of COS-7 cells transiently expressing E1, E1-R1(A/D) or E1-R3(A/D) using Annexin V-staining (right panel). The average of three independent experiments is shown. (B) Quantitative assessment of Western blot analysis of oncogenic Ras-induced Erk2 activation. NIH3T3 (left panel) and COS-7 (right panel) cells transiently expressing K-RasG12V, HA-tagged Erk2 and E1, E1-R1(A/D) or E1-R3(A/D) were subjected to western blot analysis detecting phosphorylated and total Erk2. Signals were quantified and expressed as ratio of phosphorylated and total Erk2. The ratios derived from the different conditions were normalized to EGFP, and the average of three independent experiments is shown in the figure. (C) Impact of low-affinity RBD constructs on the K-RasG12V-stimulated induction of the human MMP1-promoter. NIH3T3 cells were transiently transfected with the different RBD-constructs 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 after 24 h. The figure shows the result of three independent experiments each performed in duplicate. (D) Influence of the low-affinity RBD probes on the oncogenic Ras-stimulated invasiveness of COS-7 in a Matrigel-coated Transwell assay. The data depicted show the average from three independent experiments.