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. 2017 Jun 22;8(43):74006–74018. doi: 10.18632/oncotarget.18598

Figure 3. CBP501 attenuates EGF-dependent EMT via PI3K/Akt pathway.

Figure 3

(A) A549 cells were treated with or without EGF (50 or 100 ng/mL) in combination with CBP501 (1 or 5 μM) for 72 h. Cell lysates were analyzed by western blot assay with antibodies to E-cadherin, Vimentin, Zeb1, and β-actin. The measured band density was normalized relative to that of the control sample, with the control value set to 1. β-actin was used as a loading control. The results are the average relative intensity of three replicate blots. FL, full length; CF, cleaved fragment. (B) A549 cells were serum-starved for 1 day and then pretreated with CBP501 (1 μM) for 3 h before stimulating with EGF. The cells were treated with EGF (100 ng/ml) for 0-60 min. Cell lysates were analyzed by western blot assay (n = 3) with antibodies to pAkt, Akt, pERK1/2, ERK1/2, pSTAT3, STAT3 and β-actin. (C) Quantification of H1299 cell invasion by EGF stimulation in spheroid invasion assay. Cells were aggregated into spheroids and then induced to invade the surrounding matrix for 7 days with or without EGF (500 ng/mL) stimulation. The total area of each invading spheroid was calculated with Image-J software and taken to be a measure of cell invasion (n = 3). Red signal threshold was set to capture the total structure. Scale bar is 500 μm. Data, the mean ± SD; * and **, P < 0.05 and P < 0.005, respectively.