Fig. 2: Appearance of phospholipid peroxidation products as potential phagocytic eat-me signals in ferroptotic cells. | Cell Death & Differentiation

Fig. 2: Appearance of phospholipid peroxidation products as potential phagocytic eat-me signals in ferroptotic cells.

From: Oxygenated phosphatidylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2

Fig. 2

a HL60 cells were treated with RSL3 (1 μM, 6 h) or STS (0.25 μM, 6 h) and the gene expressions of phagocytic signals (heat shock protein HSP 90-alpha (HSP90AA1), milk fat globule-EGF factor 8 (MFGE8), growth arrest specific-6 (GAS6), intercellular adhesion molecule 3 (ICAM3), complement C1q subcomponent subunit B (C1QB), calreticulin (CALR), and V-domain Ig-containing suppressor of T-cell activation (VISR)) were detected by quantitative real-time PCR assay. Data are mean ± SEM (n = 3 biologically independent cell cultures). b The protein expressions of phagocytic signals (CALR and cluster of differentiation 47 (CD47)) and GPX4 in HL60 cells were determined by western blotting analysis. This experiment was repeated for three times. *P < 0.05 and **P < 0.01, by one-way ANOVA with LSD post hoc test. Annexin V-positive/PI-negative cells of RSL3/STS-treated HL60 cells (c) and Gpx4 KO MEFs (d) were assessed by flow cytometry. Data are mean ± SEM (n = 3 biologically independent cell cultures). ***P < 0.001 vs the control group, by one-way ANOVA with LSD post hoc test. e HL60 cells were treated with RSL3 (1 μM, 6 h) and double-stained with Liperfluo (green fluorescent) and DiD (red fluorescent). Scale = 20 μM. f HL60 cells were treated with RSL3 (1 μM) for different hours and then cocultured with THP-1 cells. The ratio of lipid peroxidation (stained with Liperfluo) was measured by flow cytometry. ***P < 0.001 vs the time (0) group, by one-way ANOVA with LSD post hoc test. Data are mean ± SEM (n = 3 biologically independent cell cultures). g Scheme for simplified network of main factors regulating ferroptosis. PUFA polyunsaturated fatty acids, PE phosphatidylethanolamines. h HL60 cells were treated with RSL3 (1 μM, 6 h) before the plasma membrane was isolated and detected by LC–MS/MS-based phospholipidomics. Data are mean ± SEM; n = 6 biologically independent cell cultures. Obtained data were displayed as volcano plots showing the changes in the levels of oxygenated phospholipids (log2 (fold change), X-axis) vs significance (−log10 (P value), Y-axis, by t-test). ox oxygenated. Each dot represents one class of phospholipids. Data of oxPEs (i) and oxPSs (j) were extracted and interpreted by principal component analysis (PCA), and the 2D score plots displayed repertoires of control and RSL3-treated cells. Ellipses: 95% confidence regions.

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