Secondary validation assays identify 15 novel hepcidin activators. (A) Workflow of the secondary assays. To validate the selected hepcidin regulators, we applied siRNA pools and 4 individual siRNAs to knockdown the genes of interest. (B) Shown are 15 genes that were validated by the experimental workflow outlined in (A). Genes were considered as validated if at least 2 of 4 siRNAs caused a significant change in hepcidin mRNA expression. In addition, we assessed whether the knockdown efficiency of each single siRNA correlated significantly with the observed changes in hepcidin mRNA expression (the correlation coefficient [r] and its significance [P value] were calculated for all obtained data points). The indicated protein functions are based on information available in the SwissProt database. (C-D) Shown are mRNA levels of hepcidin and target genes after RNAi in (C) Huh7 cells or (D) PHHs. Data for siRNA pools are shown (results for individual siRNAs are shown in supplemental Figures 2 and 3). The positive controls are indicated. Levels of mRNA expression were determined by real-time qPCR. Results are presented as fold change compared with samples transfected with scrambled siRNA. The mean of at least 3 independent experiments is shown. Significant changes are marked by *(P < .05) or **(P < .001).