Figure 6
Effects of cell migration and matrix-cell adhesion via PI3K/AKT signaling in 4T1 tumour cells.
(A) In transwell migration assay, cells were treated with or without RA-XII (100 nM), LY294002 (PI3K inhibitor) (10 μM) and RA-XII plus LY294002 for 4 hours. Representative microscopic photographs showed the stained migrated cells on the lower side of the membrane. Quantified analysis summarized the number of the migrated cells of the membrane and expressed as the percentage of the control (mean + SD of 3 independent experiments with duplicate each). (B) In fibronectin-cell and laminin-cell adhesion assays, cells were treated with or without RA-XII (100 nM), LY294002 (PI3K inhibitor) (10 μM) and RA-XII plus LY294002 for 2 hours. Cells added to the fibronectin-coated or laminin-coated wells were treated with or without RA-XII (500 nM), LY294002 (PI3K inhibitor) (10 μM) and RA-XII plus LY294002 for 2 hours. BSA coating was used as the negative control. The intensity of the coloured product from the stained bound cells determines their adhesions to fibronectin or laminin. Results were expressed as the percentage of the control (mean + SD of 3 independent experiments with duplicates each). (C) Effects of LY294002 (PI3K inhibitor), BAY11-7082 (NF-κB inhibitor) and PF-573228 (FAK inhibitor) on the expressions of the molecules involved in cell migration and adhesion in breast cancer cells. Cells were treated or without LY294002 at 20 μM, BAY11-7082 at 5 μM and PF-573228 at 1 μM for 24 hours. Cells were stained with FITC- or PE-conjugated antibodies. The histograms summarized the flow cytometric analysis from 3 to 4 independent experiments and were expressed as fold of the controls (mean + SD). Statistical differences were determined by One-way ANOVA, followed by Dunnett Test, with *p < 0.05, **p < 0.01, ***p < 0.001 against the controls. Statistical differences were also determined by unpaired Student’s t-test, with ###p < 0.001 between 2 groups. (D) Schematic diagram of the proposed underlying mechanisms of the anti-metastatic effect of RA-XII.
