Fig. 3.

Synergistic effects of EGFR TKIs combined with Akt inhibitors in parental and EGFR-TKI-resistant PC9 cells. The combined effects of EGFR inhibitors (erlotinib for PC9-ER and gefitinib for parental PC9 and PC9-GR1-5) and Akt inhibitors (GSK2141795 or AZD5363 for all cell lines) were assessed by a crystal violet viability assay performed over 8 days, b BrdU incorporation assay performed after 48 and 96 h incubation, and c apoptosis assay performed after 96 h incubation. Concentrations of drugs were chosen to be sub-inhibitory to explore the synergistic potential: erlotinib 30 µM; gefitinib 5 µM (except for PC9 where 40 nM was used); GSK2141795 (G) 2.5 µM; AZD5363 (A) 35 µM, (except for GR2 and GR5 where 1.25 and 3 µM were employed). All experiments were conducted in quadruplicates (a), or triplicates (b, c), and results are shown as mean ± SD. Asterisks indicate significant difference in ANOVA one-way test (p < 0.05) for the drug combination-treated cells compared to cells treated with either drug alone at the same time point