Figure 4: Trametinib treatment induces feedback activation of AKT and ERBB family and combination of MEK and ERBB inhibition induces synergetic suppressive effect on LUAD12C cells.

A. LUAD12C cells were treated with crizotinib (1 μM), trametinib (25 nM), or a combination of trametinib (25 nM) and crizotinib (1μM) for 48 hours. Lysates were then applied to phospho-RTK arrays. B. LUAD12C cells were treated with trametinib (25 nM) for indicated time. Lysates were subjected to immunoblotting. C. LUAD12C cells were treated with trametinib (25 nM) for 48 hours and mRNA expression level of ERBB families were determined by RT-qPCR. Experiments were conducted in triplicate and error bars represent mean ± SD. D. LUAD12C cells were treated with trametinib (25 nM) for 48 hours and mRNA expression level of ligands for ERBB families were determined by RT-qPCR. Experiments were conducted in triplicate and error bars represent mean ± SD. E. LUAD12C cells were infected with lentivirus expressing shRNAs targeting genes or non-targeting shRNA as a control, followed by selection with puromycin. Subsequently, a total 1.5 × 10⁵ of knockdown LUAD12C cells were plated in 6-well plates, and treated with DMSO or trametinib (5nM) for 10 days. Relative cell numbers were shown. Each condition was assayed in duplicate determinations in 2 independent experiments and error bar represent mean ± SE. F. Lysates from knockdown LUAD12C cells after selection were subjected to immunoblotting.