Fig. 2. Adverse synergy between maternal iron excess and inflammation targets placental and embryo endothelium to cause its apoptosis.

a–d Maternal systemic inflammation was induced in iron-adequate (gray circles) and iron-loaded (blue circles) dams on E15.5 by a single subcutaneous injection of 0.5 μg/g LPS for 24 h. Western blot (left) and quantitation (right) of a whole placenta and b embryo liver for apoptotic marker cleaved caspase-3 normalized to β-actin (n = 3-9/group). c TUNEL stain of placental sections. Representative images of n = 3 sections/group. d Immunohistochemistry for cleaved caspase-3 (brown) and endothelial marker CD31 (red) in paraffin-embedded placenta (n = 3), embryo lung (n = 5), heart (n = 5), and liver (n = 5) sections. Scale bar= 50 μm. a–d Embryo and placentas were randomly selected for analysis. e Primary HUVECs were treated with 100 μM ferric ammonium citrate (FAC) for 24 h prior to being stimulated with IL6, IFNγ, IL1α, IL1β, or TNFα (all 50 ng/ml) in solvent or FAC-supplemented media for 16 h. Western blot (left) and quantitation (right) for cleaved caspase-3 normalized to β-actin, representative image of n = 4 independent experiments. Ferritin-H is a marker of cellular iron loading. a, b, e Error bars represent mean ± s.e.m. Statistical differences were determined by two-way ANOVA (denoted by &) or one-way ANOVA (denoted by #) followed by Holm-Sidak method for multiple comparisons. P-values are indicated in each figure panel. Source data are provided as a Source data file.