Fig. 5

ICA regulated autophagy and apoptosis by acting on AMPK. A, B Western blotting showing the expression of AMPK, P62, and LC3B proteins in MDA-MB-468 and 4T1 after ICA with or without MET (5 mM) exposure, with β-actin as an internal control. C, D MDA-MB-468 and 4T1 cells were treated with ICA (25 µM) with or without MET (5 mM). Apoptosis was analyzed using flow cytometry. E, F The protein expression levels of P62, LC3B, as well as AMPK, mTOR, ULK1, and their phosphorylated proteins were detected using western blotting analyses after knocking down AMPK in TNBC cells. G, H The apoptosis was analyzed by flow cytometry after knocking down AMPK in TNBC cells. Bars represent the means ± SD of at least three independent experiments; ns, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001. ICA icariin, TNBC triple-negative breast cancer, MET metformin, P62 SQSTM1, BECN1 Beclin1, LC3B MAP1LC3B, AMPK adenosine 5ʹ-monophosphate (AMP)-activated protein kinase, mTOR mammalian target of rapamycin, ULK1 Unc-51-like kinase 1