Fig. 5: Ferroptotic supernatant exposure enhances macrophage cytokine release.

A Schematic of the supernatant transfer strategy from GPX4 control and GPX4 KO SCLC cells 24 h after Fer-1 withdrawal to iBMDMs. After supernatant transfer iBMDMs were incubated with ctrl. or KO supernatants for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. B iBMDMs were incubated with ctrl. (GPX4 ctrl. SN) or KO supernatants (GPX4 KO SN) from SCLC cells for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. TNF-alpha was quantified using ELISA. C iBMDMs were incubated with ctrl. or KO supernatants from SCLC cells for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. IL-6 was quantified using ELISA. D Differentiated pBMDMs were treated with ctrl. or KO supernatants for 6 h IFN gamma [25 ng/ml], LPS [10 ng/ml], and Brefeldin A (BFA) [5 µg/ml]. Combined CD11b⁺, F4/80⁺, TNFα⁺ cells within live cells were quantified using flow cytometry. E pBMDMs were incubated with ctrl. or KO supernatants for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. TNF-alpha was quantified using ELISA. F pBMDMs were incubated with ctrl. or KO supernatants for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. IL-6 was quantified using ELISA. G iBMDMs were incubated with ctrl. or KO supernatants for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. Dot Blot analysis on the supernatants was performed using the cytokine profiler mouse XL cytokine array (R&D Systems). H iBMDMs were incubated with supernatants from parental Pfa1 or Pfa1 MEFs with stable FSP1 overexpression [9] (mFSP1 OE) as indicated for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. TNF-alpha was quantified using ELISA. I iBMDMs were incubated with standard ctrl. or KO supernatants or respective boiled “cooked” supernatants as indicated for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. TNF-alpha was quantified using ELISA. J iBMDMs were incubated with empty vector (EV) or supernatants from ZBP1 induced (ZBP1 OE) + emricasan [2.5 µM] for 6 h with IFN gamma [25 ng/ml], after which LPS [10 ng/ml] was added for a total of 24 h. IL-6 was quantified using ELISA. All schemes were created with BioRender.com. Data information: B–F, H–J Graphs show data of means ± SEM of at least 3 independent experiments. One- or two-way ANOVA was used to calculate p-values. ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Source data are available online for this figure.