Fig. 3

ICA induced cell cycle arrest, apoptosis, and autophagy in TNBC cells. A, B MDA-MB-468 and 4T1 cells were treated with different concentrations of ICA (0, 12.5, and 25 µM). The cell cycle distribution was analyzed using flow cytometry. C, D MDA-MB-468 and 4T1 cells were treated with ICA (0, 12.5, and 25 µM) followed by Annexin V/PI double staining and analyzed using flow cytometry. E MDA-MB-468 and 4T1 cells were treated with ICA (25 µM) and detected by TEM. The red arrows represent autophagosomes, and the black arrows denote autolysosomes. F, G MDA-MB-468 and 4T1 cells treated with different concentrations of ICA (0, 12.5, and 25 µM) were stained with MDC and imaged using a microscope (20 ×). The green circles represent autophagosomes. H, I The protein expression of P62, BECN1, and LC3B was determined using western blotting with β-actin as an internal control. Bars represent the means ± SD of at least three independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group (0 µM). ICA icariin, TNBC triple-negative breast cancer, TEM transmission electron microscope, P62 SQSTM1, BECN1 Beclin1, LC3B MAP1LC3B