Fig 2.

Effects of combination of EGFR inhibitors with cMET inhibitor EMD 1214063 on soft agar assay. Cells were suspended in complete RPMI medium containing 0.3% soft agar and seeded in triplicate on 35-mm dishes precoated with 0.6% agar in complete growth medium mixed with corresponding drug and incubated at 37°C, 5% CO₂. After 10 days, colonies were photographed and counted in 10 randomly chosen fields and expressed as means of triplicates. For staining of the colonies, 0.005% crystal violet in 20% methanol solution were added to the dishes for 30 minutes and washed until the background becomes clear. Drug concentrations were as follows; Gefitinib 1uM, EMD 121463 5uM, and Cetuximab 200 ug/ml, in MDA-MB-468; Gefitinib 5uM, EMD 121463 5uM, and Cetuximab 200 ug/ml in MDA-MB-231. Colonies were not formed in HCC 1395 and there were no difference between single agent and combination in T47D (data not shown). The data are mean ± standard deviations of triplicates (*, P<0.001 vs EMD 121463; **, P<0.05 vs EMD 121463; ***, P<0.001 vs gefitinib; #, P>0.05 vs cetuximab).