Fig. 4: Exposure to ferroptotic supernatants induces transcriptional reprograming of macrophages.

A pBMDMs were incubated with supernatants from GPX4 control (GPX4 ctrl SN) or GPX4 KO (GPX4 KO SN) SCLC cell lines (Bebber et al. [35]) at day 7 post-differentiation. CD11b⁺, F4/80⁺ cells were gated within live cells. Representative Flow Cytometry analysis is shown. B Percentage of combined CD11b⁺, F4/80⁺ pBMDMs within live cells was plotted. C Schematic of supernatant transfer strategy from Pfa1 to pBMDMs (n = 3 per condition). D Differentiated pBMDMs incubated for 24 h with supernatants as depicted in C were subjected to RNA sequencing. The 25 most upregulated and downregulated genes are shown. The genes are clustered row-wise by expression patterns using z-scores of normalized expression. E Positively enriched immune system-related GO terms in pBMDMs exposed to ferroptotic supernatants were chosen within the top 100 GO terms enriched. F pBMDMs were subjected to the indicated supernatants from Pfa1 MEFs for 24 h. The indicated transcripts were quantified by qPCR. G iBMDMs were incubated with supernatants from live (supernatant collected after 72 h from Pfa1 MEFs -4OHT) or ferroptotic Pfa1 MEFs (supernatants collected after 72 h from Pfa1 MEFs +4OHT) for 24 h. Surface expression of CD14 was quantified by antibody-based staining and flow cytometry. Mean fluorescence intensity (MFI) is shown. H RAW264.7 cells were incubated, stained, and analyzed as in (G). All schemes were created with BioRender.com. Data information: B, F, G Graphs and D heatmap show data of means ± SEM of 3 independent biological replicates. 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.