Holo-transferrin triggers induction of hepcidin, Tfr2, and ferritin in Tfrc^Alb-Creand Tfrc^fl/flhepatocytes. Primary hepatocytes were isolated from livers of Tfrc^Alb-Cre mice and control Tfrc^fl/fl mice. The cells were cultured in serum-free media and left untreated or treated overnight with 30 μM apo- or holo-transferrin. At the end point, the cells were harvested and used for preparation of RNA and protein lysates. (A) qPCR analysis of Hamp mRNA. (B) Western blot analysis of Tfr1, Tfr2, ferritin, β-actin, pSmad5, Smad1, pStat3, and Stat3. (C) qPCR analysis of Il6 mRNA. Data in (A) and (C) are mean ± standard error of the mean. Statistically significant differences across genotypes are indicated by P values (or ns [nonsignificant]) and across treatment (no treatment vs apo- or holo-transferrin) by ***P < .001, 2-way ANOVA.