Figure 6.

Effects of the combined platelet-activating factor receptor (PAFR) and epidermal growth factor receptor (EGFR) inhibition in CASKI cells. (A) CASKI cells were treated with cetuximab (100 µg/ml) and/or WEB 2086 (50 µM) for 24 h. Cell death was evaluated by propidium iodide staining, followed by flow cytometry. Cells with hypodiploid DNA content (sub-G1 peak) were considered apoptotic cells. Representative histograms of three independent experiments. (B) For the MTT assay, CASKI cells were seeded at 2,500 cells/well in 96-well cell culture plates. Then, cells were treated with cetuximab (100 μg/ml) and/or WEB 2086 (100 μM) for 72 h in medium supplemented with 2% fetal bovine serum (FBS). After treatment, cells were incubated with a solution of MTT. The formazan crystals generated were solubilized in DMSO and the optical density was measured at a wavelength of 538 nm. Cell viability was expressed as a percentage of the control. Values represent mean + SD of four independent experiments. (C, D) For the clonogenic assay, CASKI cells were seeded at low density and treated for 13 days with cetuximab (100 µg/ml) and/or WEB 2086 (50 µM). Then, colonies were stained with crystal violet and photographed. (C) Representative image of the colonies of the CASKI cell line after targeted therapies against EGFR and PAFR. (D) Crystal violet was eluted in methanol and absorbance at 595 nm was measured. Values represent mean + SD of five independent experiments. One-way ANOVA and Tukey’s post-test were used. *P < 0.05, **P < 0.01, ***P < 0.001.