Fig. 1

Protective effect of BAZ against erastin-induced ferroptosis in HT22 cells. A. Chemical structure of bazedoxifene (BAZ). B, C. Cytotoxicity of erastin alone (B) and its protection by BAZ (C) after 24-h treatment with erastin ± BAZ at indicated concentrations (MTT assay, n = 5). D. Calcein-AM/PI double staining of live and dead cells following 8-h treatment with 0.8 μM erastin ± 1 μM BAZ (fluorescence microscopy images, scale bar = 60 μm). E‒G. mRNA levels of GPX4 (E), PTGS2 (F) and ACSL4 (G). Cells were treated with 0.8 μM erastin ± 1 μM BAZ for 2 h (for detection of PTGS2) or 8 h (for detection of GPX4 and ACSL4), and then their mRNA levels were determined by RT-qPCR (n = 3). H. Cellular levels of GPX4, COX2 and FACL4 proteins after 8-h treatment with 0.8 μM erastin ± 1 μM BAZ (Western blotting). I‒K. Cellular levels of NO (I), ROS (J) and lipid-ROS (K) after 8-h treatment with 0.8 μM erastin ± 1 μM BAZ (analytical flow cytometry). The left panels of I, J, K are the histograms, and the right panels are respective quantitative intensity values (n = 3). L. Cellular levels of lipid-ROS after 8-h treatment with 0.8 μM erastin ± 1 μM BAZ. The cells were stained with BODIPY and Hoechst, and then subjected to confocal microscopy (scale bar = 10 μm). M. Levels of mitochondrial ROS after 8-h treatment with 0.8 μM erastin ± 1 μM BAZ. The cells were stained with MitoSOX, MitoTracker and Hoechst, and then subjected to confocal microscopy (scale bar = 10 μm). Quantitative data are presented as mean ± S.D. (* or ^# P < 0.05; ** or ^## P < 0.01)