Fig. 3

Identification of ACTN4 as the direct target of EA in breast CSCs. a MDA-MB-231 CSCs were treated with EA at varying concentrations, a 100-kDa protein band was protected from digestion in a dose-dependent manner. The arrowhead indicates the 100-kDa protein; b Mass spectrogram identified the 100-kDa-targeted protein of EA as ACTN4; c Amino acid sequence of actinin-4. Bold letters: peptides that correspond to peaks identified by mass spectrometry; d EA administration significantly led to an average suppression in the ACTN4 expression levels, accompanied by inhibited expressions of β-catenin and vimentin in six tumors from EA-treated MMTV-PyMT transgenic mice tumors. A quantitative measurement was conducted to analyze with Image J (**P < 0.01, values represented as the mean ± SD, n = 6). Cell lysates of Lanes 1, 3, 5, 7, 9 and 11 came from six MMTV-PyMT transgenic mice in vehicle groups, while proteins of Lanes 2, 4, 6, 8, 10 and 12 belonged to six mice in EA treatment groups; e EA inhibited ACTN4 expression in the MDA-MB-231, BT-549, and MCF-7 cells by western blot analysis