Fig. 2: PQS promotes iron-dependent lipid peroxidation to induce ferroptosis in macrophages.

A CCK-8 assay for cell viability in RAW264.7 macrophages pretreated with different cell death inhibitors—Fer-1, NEC, VAD, DIS, and 3-ME—for 3 h and then treated with 10 μg/mL PQS for 24 h. Data represent the means ± SD (n = 3, one-way ANOVA with Tukey’s multiple comparisons test). B Quantification of total iron in RAW264.7 macrophages treated with PQS or PQS + Fer-1; macrophages treated with Ferric ammonium (FA) and DMSO served as the positive and negative controls, respectively. Data represent the means ± SD (n = 3, one-way ANOVA with Tukey’s multiple comparisons test). Quantification of total iron (C) and CCK-8 assay for cell viability (D) in RAW264.7 macrophages treated with or without PQS in RPMI1640 + FBS medium (normal iron supplemented by FBS) and iron-free RPMI1640 medium without FBS. Data represent the means ± SD (n = 3, two-way ANOVA with Tukey’s multiple comparisons test). E Representative images of Phen Green fluorescence staining to label labile free iron in RAW264.7 macrophages treated with PQS or PQS + Fer-1; macrophages treated with FA served as the positive control. Phen Green: green; Hoechst nucleus: blue. Scale bar = 20 µm. F Representative images of C11-BODIPY fluorescence staining to label lipid ROS in RAW264.7 macrophages treated with PQS or PQS+Fer-1; macrophages treated with ERA served as the positive control. C11-BODIPY: green; Hoechst nucleus: blue. Scale bar = 20 µm. G Flow cytometry for measurement of lipid ROS with 5 μM C11-BODIPY lipid probe in RAW264.7 macrophages treated with PQS or PQS + Fer-1; macrophages treated with ERA and DMSO served as the positive and negative control, respectively. H Flow cytometry for measurement of intracellular ROS with 10 μM DCF probe in RAW264.7 macrophages treated with PQS or PQS+Fer-1; macrophages treated with ERA and DMSO served as the positive and negative control, respectively. I CCK-8 assay for cell viability in RAW264.7 macrophages pretreated with different ferroptosis inhibitors, Lip-1, Trolox, and VK2, for 3 h and then treated with IC₅₀ (PQS) for 24 h; macrophages treated with DMSO served as the negative control. Data represent the means ± SD (n = 3, one-way ANOVA with Tukey’s multiple comparisons test). J Flow cytometry for measurement of mitochondrial ROS with MitoSOX probe in RAW264.7 macrophages treated with PQS or PQS + Fer-1; macrophages treated with ERA and DMSO served as the positive and negative controls, respectively. Source data are provided as a Source Data file.