Fig. 3.

OA abrogates the cytotoxic effect of EGFR-TKIs on EGFR-sensitive NSCLC cell lines (a) Morphology of the indicated NSCLC cell lines after vehicle (DMSO, NT), Gefitinib (100 nM or 20 μM in H1975 cells), or Gefitinib (100 nM) + OA (100 μM) exposure was assessed. For each experiment, five images of random fields were acquired. A representative image is shown. b Left panel, 800 surviving cells per well were seeded in 6-well plates, followed by incubation for 10 days until a mass of cells was visible to the naked eye. The synchronized cell masses were exposed to vehicle, Gefitinib, or Gefitinib + OA for 48 h and stained with crystal violet. Right panel, the number of colonies was assessed using ImageJ software. p values were determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Data represent the mean ± SD of three replicate determinations. (c) Upper panel, apoptotic rates of the indicated NSCLC cell lines were assessed by flow cytometry after vehicle, Gefitinib, or Gefitinib + OA treatment for 48 h. Lower panel, apoptotic rates were quantified. The results represent data from three independent experiments. p values were determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. d Upper panel, apoptotic rates of the indicated NSCLC cell lines were assessed by flow cytometry after vehicle (DMSO, NT), Gefitinib (100 nM or 20 μM in H1975 cells), Gefitinib (100 nM) + OA (100 μM), or Gefitinib (100 nM) + OA (200 μM) treatment for 48 h. Lower panel, apoptotic rates were quantified. The results represent data from three independent experiments. p values were determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. e The indicated cell lines were exposed to vehicle, Gefitinib, or Gefitinib + OA for 48 h. The cells were then harvested for Western blotting to detect the indicated signaling proteins (upper panel) and apoptotic proteins (lower panel). A representative blot is shown from three independent experiments. Right panel, the quantitation of western bolt