Fig. 2

EA inhibits breast cancer cell proliferation, migration, and invasion abilities associated with CSC limitation. (A) EA suppressed the proliferation of the breast cancer cell lines MDA-MB-231, BT-549, and MCF-7, while posing little inhibitory effects on the human normal mammary epithelial cell MCF-10A (**P < 0.01 versus control, values represented as the mean ± SD, n = 3) at different dose- and time-intervals indicated; (B) The wound healing and chamber invasive assay revealed that breast cancer cell migration and invasion were inhibited by EA in a time- and dose-dependent manner (**P < 0.01 versus control at 12 h, ^# P < 0.05, ^## P < 0.01 versus control at 24 h, ^&& P < 0.01 versus control at 48 h, values represented as the mean ± SD, n = 3); (C) EA dose-dependently reduced the CSC populations in the breast cancer cells. Representative dot plots of CD44⁺CD24^−/low cell surface markers in the breast cancer cells MDA-MB-231 and BT-549 (**P < 0.01 versus control, values represented as the mean ± SD, n = 3); (D) EA limited the primary and secondary CSC mammosphere of the MDA-MB-231 and BT-549 cells in a dose-dependent manner (*P < 0.05, **P < 0.01, values represented as the mean SD, n = 3); (E) a. Western blotting analysis validated that EA elevated p-β-catenin, while inhibiting p-GSK-3β and p-AKT in the MDA-MB-231 stem-like cell lysates time-dependently; b. EA did not suppress the β-catenin mRNA level after 24 h administration; c. EA inhibited its transcriptional activity as detected by the TCF/LEF luciferase assay (**P < 0.01, values represented as the mean ± SD, n = 3); d. The relative mRNA expressions of the β-catenin downstream genes were inhibited by EA in the MDA-MB-231 stem-like cell lysates (*P < 0.05, **P < 0.01, values represented as the mean ± SD, n = 3)