Figure 4. Ferroptosis is the underlying mechanisms of αMSH particle-induced cell death.

a, Quantification of cell death (Sytox green +) in MCF10A cells cultured in full media (Full) or amino acid-starved (AA-st) conditions in the presence or absence of 15μM αMSH-PEG-C′ dots and (a) 1μM Ferrostatin-1 (Fer-1) after 40 hours. Error bars indicate mean +/− standard deviation. N=3 per group. Each replicate is from one biological experiment, quantified with five independent fields of view. b, Quantification of cell death (Sytox green +) in MCF10A cells cultured in full media (Full) or amino acid-starved (AA-st) conditions in the presence or absence of 15μM αMSH-PEG -C′ dots and 50νM butylated hydroxyanisole (BHA), 200νM ascorbic acid (Asc Acid), 100νM Trolox, 10mM N-acetylcusteine (NAC), or 5mM glutathione (GSH) after 40 hours. Error bars indicate mean +/− standard deviation. N=5 per group. Each replicate is from one biological experiment, quantified with five independent fields of view. c, Lipid ROS accumulate prior to death in cells treated with 15μM αMSH-PEG-C′dots and amino acid withdrawal. Images show treated cells cultured in the presence of C11-BODIPY that detects lipid ROS. Note that the fluorescence intensity of C11-BODIPY increases several hours before cell death (times indicated on each image prior to cell death in bottom image). Scale bar=10μm. Graph: quantification of C11-BODIPY fluorescence in particle-treated and amino acid-starved cells (red line), or erastin-treated cells (black line). Mean intensities out of five cells +/− standard deviation are shown from one biological experiment. Time zero indicates the time of cell death determined by DIC microscopy. Note that C11-BODIPY staining increases in intensity between three and four hours prior to cell death. d, Quantification of cell death (Sytox green +) in MCF10A cells cultured in full media (Full) or amino acid-starved (AA-st) conditions in the presence or absence of 15μM αMSH-PEG-C′ dots and 100μM deferoxamine (DFO). Error bars indicate mean +/− standard deviation. N=3 per group. Each replicate is from one biological experiment, quantified with five independent fields of view. e, Images from time-lapse analysis of MCF10A undergoing ferroptosis in amino acid-starved conditions with 15μM αMSH-PEG-C′ dots. Note that death (Sytox green positivity) spreads cell-to-cell from the left side of the image to the right. Right image shows the position of each dead cell nucleus pseudocolored to represent the relative timing of cell death. Scale bar=10μm.