Figure 3

Effect of absorbed AP components from 800, 1600 and 3200 μg/ml APW on NFκB expression and the expression of metastasis-related genes. (A) NFκB ELISA assay. The OD values were normalised with the corresponding protein concentrations. Each column represents the mean + S.D. in duplicates of three independent experiments. (B) Western blot analysis of p-NFκB and NFκB expression. Representative immunoblots showing the effects of absorbed AP components from 800, 1600 and 3200 μg/ml APW on EC-109 cellular expression of p-NFκB and NFκB. Actin expression was determined to confirm equal protein loading. (C) The histogram showed quantified results of protein levels, which were adjusted with corresponding β-actin protein level and expressed as folds of control (mean fold of control + S.D. of three independent experiments). Differences between the treatment groups and the control group were determined by one way ANOVA followed by post-hoc Dunnett’s test, *p < 0.05, ***p < 0.001 as compared with vehicle control. (D–G) Real-time PCR showed the expression of metastasis-related genes. EC-109 cells were treated with absorbed AP components from 800, 1600 and 3200 μg/ml APW for 24 h and then the cells were collected for RNA extraction. Quantitative real time PCR analyses of mRNA of TM4SF3, MMP9, HER2 and CXCR4 genes were shown. Data were normalized to corresponding GAPDH expressions as internal control. The results of mRNA expressions are expressed as fold of control (mean fold of control + S.D. from 3 independent experiments). Differences among all groups were determined by one-way ANOVA followed by post-hoc Tukey’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001 as compared with vehicle control.