Fig. 10: Exogenous 4HNE inhibits macrophage-mediated killing and induces host cell death.

a 4HNE release of BMDMs during C. albicans infection. Twelve hours post infection. N = 3 in duplicate. Two-tailed Mann–Whitney Test. Box-and-whisker plots indicating median, 25th/75th percentiles, and the minimum/maximum values. b Macrophage-mediated killing of cells incubated with 4HNE. MOI 1:5. 12 h post infection. N = 3 in triplicate. Ordinary one-way ANOVA corrected for multiple comparison. Box-and-whisker plots indicating median, 25th/75th percentiles, and the minimum/maximum values. c Representative images of C11 oxidation of BM-derived macrophages incubated for 4 h with 50 μM 4HNE. Blebbing (apoptotic cells) indicated with arrows. Scale bar 50 (left) and 10 μm (right). Dotted squares indicate magnified areas on the right. N = 3 in triplicate. Quantification of d ferroptotic (C11ox) and e apoptotic (blebbing) macrophages. N = 3 in triplicate. Two-tailed Mann–Whitney test. Box-and-whisker plots indicating median, 25th/75th percentiles, and the minimum/maximum values. f Representative immunoblot of MLKL phosphorylation. BM-macrophages were treated with 50 μM 4HNE for 4 h. Cell lysates were separated via SDS-PAGE and probed for pMLKL, total MLKL, and β-actin. N = 4 independent experiments. g Representative immunoblot of gasdermin-D cleavage. BM-macrophages were infected with C. albicans (MOI 10) for 2 h followed by incubation with 50 μM 4HNE for 4 h. Cell lysates were separated via SDS-PAGE and probed for GSDMD, and β-actin. N = 4 independent experiments.