Fig. 3

Mitochondrial pathway plays an important role in the enhancing effect of FZKA on cell apoptosis by Gefitinib. a Caspase-9 activity was dramatically increased in the combined group treated with FZKA and Gefitinib. Caspase-9 activity assay was done in the lung cancer cells (A549 and PC9) treated with FKZA, Gefitinib, or FZKA combined with Gefitinib for 24 h, respectively. ***p < 0.0001; one-way ANOVA. Data represent mean ± SD of three independent experiments. b, c Both cleaved-Caspase-9 and Cyt-C, two critical proteins in the mitochondrial apoptotic pathway, were the most expressed in the combined group treated with FZKA and Gefitinib. Western blot was performed to detect the cleaved Caspase-9 and Cyt-C expression in lung cancer cells (A549 and PC9) treated with FKZA, Gefitinib, or FZKA combined with Gefitinib for 24 h, respectively. GAPDH was used as a loading control. Densitometric analysis was performed using ImageJ. d Mitochondrial membrane potential (MMP) was reduced the most in the combined group treated with FZKA and Gefitinib. MMP assay was carried out in lung cancer cells (A549 and PC9) treated with FKZA, Gefitinib, or FZKA combined with Gefitinib for 24 h, respectively. Representative photos were shown as indicated (top panel). And the ratios of J-aggregates/monomer were shown as column diagrams (bottom panel). The red fluorescence means J-aggregates when the MMP is high, and the green fluorescence means monomer when the MMP is low. ***p < 0.0001; one-way ANOVA. Data represent mean ± SD of three independent experiments