Liver endothelial expression of metal-ion transporter Slc39a8 (encoding ZIP8) is not regulated by dietary iron. (A-B) Three-week-old wild-type 129S6 male mice were fed a purified iron-deficient diet (2-6 ppm iron as background iron), iron-sufficient diet (37 ppm iron as ferric citrate), or high-iron diet (10 000 ppm carbonyl iron) for 4 weeks (n = 3-4/diet). Liver endothelial cells were isolated by fluorescence-activated cell sorting and analyzed by (A) mass spectrometry–based quantitative proteomics for abundance of ZIP8 protein or (B) RNA-seq for Slc39a8 mRNA expression in counts per million (CPM). Only cells from the iron-sufficient and high-iron diet groups were analyzed by RNA-Seq. (C-F) Seven-week-old C57BL6/J female mice were fed a purified iron-deficient diet for 1 week, then switched to a high-diet (20 000 ppm carbonyl iron) for 18 hours (n = 6/diet). Endothelial cells were isolated from livers by magnetic-activated cell sorting and analyzed for (C) Tfrc or (D) Bmp6 relative to Rpl19 mRNA expression by qRT-PCR, (E) ferritin heavy chain 1 (FTH1) and ZIP8 protein expression relative to ß-actin by western blot and chemiluminescence quantitation, and (F) Slc39a8 expression relative to Rpl19 by qRT-PCR. Bar graphs represent mean ± SEM, with individual points indicating the number of animals. Statistical differences between groups were determined by the 2-tailed Student t-test for normally distributed values or the Mann-Whitney U test for nonnormally distributed values. ns, not significant.