Fig. 4. Maternal iron loading promotes oxidative stress in placental endothelial cells.

a Iron-adequate and iron-loaded dams were treated with solvent or LPS (0.5 µg/g) on E15.5 for 6 h. Placentas from solvent and LPS-treated mice were collected for endothelial cell isolation by magnetic separation using Cd31 beads and total RNA was analyzed by RNA-Seq. b Cd31 expression relative to Rpl4 in the Cd31-unbound (non-endothelial cells) and Cd31-bound (endothelial cells) fractions. c Endothelial cell expression of iron importer Tfrc relative to Rpl4 from iron-adequate (light gray circles) and iron-loaded (dark blue circles) placentas. b, c Placenta cells isolated from n = 8 dams. d, e RNA-Seq Ingenuity Pathway Analysis of significantly downregulated (white bars) and enriched (red bars) genes (Z-score < and > than ±1.5) from placental endothelial cells comparing hepcidin KO and WT mice after d solvent and e LPS injection. f–h Primary HUVECs were treated with solvent (gray circles), or different forms of iron (light blue circles) including ferric ammonium citrate (100 µM, FAC), hemin (20 µM), or holo-ferritin (Ft, 2 mg/ml) for 40 h and analyzed for f TFRC g NQO1 and h HMOX1 expression. n = 3 independent experiments. b, c, f–h Error bars represent mean ± s.e.m. Statistical differences were determined by two-tailed Mann–Whitney U, two-tailed Student’s t-test (denoted by *, P-values are indicated in each figure panel), or one-way ANOVA followed by Holm-Sidak method for multiple comparisons (denoted by ^#P < 0.05, ^###P < 0.001, ^####P < 0.0001). Source data are provided as a Source data file.