Fig. 4

ACTN4 overexpression abrogated the anti-cancer and anti-metastatic effects induced by EA on breast cancer. a MDA-MB-231, BT-549 and MCF-7 cells were transfected with ACTN4 recombinant plasmids and treated with or without 25 μM EA for 24 h. LY294002 (10 μM) was used as a positive control for this assay. The findings revealed that ACTN4 overexpression enhanced breast cancer cell number, and effectively restored the inhibition effects of EA on breast cancer cells (*P < 0.05, **P < 0.01, values represented as the mean ± SD, n = 3); b ACTN4 overexpression endowed EA-treated cells with increased migratory and invasive ability (**P < 0.01, values represented as the mean ± SD, n = 3). LY294002 (10 μM) was used as a positive control for this assay; c ACTN4 overexpression abrogated the CSC suppressive effects induced by EA, presenting as increased CSC proportion and enhanced sphere formation capability (**P < 0.01 EA + empty vector group versus EA + ACTN4 group in 1st CSC sphere generation, ^## P < 0.01 EA + empty vector group versus EA + ACTN4 group in 2nd CSC sphere generation, values represented as the mean ± SD, n = 3)