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. 2019 Dec 2;12:10547–10558. doi: 10.2147/OTT.S225918

Figure 5.

Figure 5

DHA sensitizes PC-9 cells to gefitinib by cholesterol displacement. (A–B) PC-9 cells were incubated in serum-free medium with or without cholesterol (10μM) for 2hrs at 37°C. Then, PC-9 cells were incubated with the indicated concentration of gefitinib (1μM), DHA (25μM) or combined treatment for 24hrs. Before the collection of cell lysates and immunoblot analysis, cells were stimulated with 50ng/mL EGF for 20 min at 37°C. EGFR, and its phosphorylated protein expression were detected by Western blot as described. Bars represent SEM, *p<0.05, ***p<0.001 versus non-treated control of PC-9 cells. (C–D) Inhibition rate of PC-9 cells was assessed after 24hrs of stimulation of combination of gefitinib, cholesterol (10μM) and DHA (25μM) by CCK8 assay. Bars represent SEM, ***p<0.001 versus non-treated control of PC-9 cells. (E–F) PC-9 cells were stained with Annexin V and PI and apoptosis cells were quantitated by flow cytometer after cholesterol (10μM), DHA (25μM) and gefitinib (1μM) treatment or combined treatment as described above. Results from the experiments are shown as means ± SEM. The data are presented as the percentage of cell inhibition rate to unstimulated cells (0μM). Bars represent SEM, *p<0.05, ***p<0.001 versus non-treated control of PC-9 cells. ## p<0.01 versus non-DHA-treated of PC-9 cells. (G) PC-9 cells were incubated with indicated concentration of DMSO, DHA (25μM) for 24hrs, cholesterol (10μM), then exposed for 3hrs to fluorescent probe with gefitinib skeleton (10μmol/L). Confocal images of cells show fluorescence of DAPI in blue, fluorescent probe in green, and are merged in Lane 3.