Fig. 4

PGRMC1 promotes ferroptosis via lipophagy. A and B Lipid droplets in HN4 parental cells and PCC with or without erastin treatment. HN4 cells were transfected with vector (vtr) or PGRMC1 overexpression (O/E) vector or treated with 100 nM progesterone (P4). HN4PCC were transfected with vector or shPGRMC1 or treated with 20 μM AG205, a PGRMC1 antagonist. The cells were treated with or without 30 nM Wortmannin plus DMSO or 10 μM erastin for 24 h. Lipid droplets (green) were quantified using ImageJ, displayed as a heatmap relative to PCC vector control. Scale bar 10 μm. C Immunoblotting in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin treatment for 24 h. D Co-staining of Lysotracker™ Deep Red and LC3-GFP (green) in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin for 4 h. Nuclei (blue) were stained with DAPI. Scale bar 10 μm. E and F Quantification of cellular lipid contents by gas chromatography and mass spectrometer (GC-MS) and of free fatty acids in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition. The GC-MS data were normalized to HN4 vector control. O/E, PGRMC1 overexpression vector; sh, shPGRMC1; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data are means and s.d. from three technical replicates. ***P < 0.001 relative vector control