Figure 7.

Anomanolide C induces ferroptosis is related to autophagy activation in TNBC cells. (A-B) ROS formation in the absence or presence of AC combine with autophagy inhibitor, 3-MA (1 mM) was observed by flow cytometry. Quantification of ROS levels and representative images are shown. (C-D) Immunofluorescence analysis of Fe²⁺ levels in MDA-MB-231 and BT549 3D spheroids treated with or without AC and 3-MA for 24 h. Quantification of immunofluorescence analysis were shown. Scale bar, 20 μm. (E, G) MDA-MB-231 and BT549 cells co-treated with or without AC and 3-MA for 24 h were collected and lysed, and the expression of p62, LC3, FTH1 and ACSL4 was analyzed using immunoblotting. beta-actin was used as a loading control. (F, H) Quantification of immunoblotting analysis were shown. (I) BT549 cells co-treated with or without AC and ferroptosis inhibitor, Fer-1 for 24 h were collected and lysed, and the expression of p62, LC3, FTH1 and ACSL4 was analyzed using immunoblotting. beta-actin was used as a loading control. (J) Quantification of immunoblotting analysis were shown. Data are presented as the mean ± SEM. These results are consistent with those of at least three different experiments. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. Statistical significance was determined relative to the appropriate AC groups.