Figure 1.

Inhibition of mutant ^V600EB-Raf reduces the metastatic potential of melanoma cells. A, siRNA-mediated reduction of mutant ^V600EB-Raf protein expression persists for 8 to 10 days in melanoma cells. SiRNA-mediated knockdown of B-Raf reduces protein expression in cells for 8 to 10 days following nucleofection compared with controls nucleofected with buffer or scrambled siRNA. Erk 2 and α-enolase were used as controls for protein loading. B, siRNA-mediated reduction of mutant ^V600EB-Raf protein decreased formation of melanoma metastases in the lungs of nude mice. SiRNA against B-RAF or scrambled siRNA was introduced into GFP-tagged 1205 Lu cells; 36 hours later, cells were i.v. injected into nude mice. Photographs show presence of GFP-tagged tumors in the lungs of mice 17 days later. Control cells were nucleofected with buffer only or scrambled siRNA. SiRNA mediated down-regulation of B-Raf protein expression reduced tumor number (C), area (D), and large-sized metastases (E) in the lungs of nude mice. Number of tumors and area occupied by GFP-tumors were quantified in a minimum of six fields per lung from 5 to 10 animals. Tumors were grouped into two pixel-based size ranges (<1,500 or >1,500 pixels). F, siRNA-mediated reduction of mutant ^V600EB-Raf expression (activity) in UACC 903M cells decreased formation of lung metastases. SiRNA against ^V600EB-RAF or a scrambled siRNA control were introduced into GFP-tagged UACC 903M cells; 36 hours later, cells were i.v. injected into nude mice, and 17 days later, lung metastases were quantified. Numbers of tumors within particular size ranges (<1,500 or >1,500 pixels) were quantified in a minimum of six fields per lung from 5 to 10 animals.