High-throughput siRNA screening identifies SMAD7 as a suppressor of hepcidin expression. (A) Schematic representation of the high-throughput siRNA screening. Huh7 cells were seeded in 384-well plates containing siRNAs from the ThermoFisher siGenome siRNA library (Dharmacon) as well as siRNAs directed against HFE and SMAD4 as positive controls and scrambled siRNAs as a negative control. Twenty-four hours later, cells were transfected with a firefly luciferase reporter construct containing the 2.7-kb human hepcidin promoter (WT_2.7kb) and a renilla luciferase control vector. Dual luciferase activities were measured 48 hours later. (B) The results of the knockdown of SMAD4, HFE, SMAD7, JAK1, and BMPR1A (ALK3) genes are presented as a mean of the z-scores from 2 replicates. (C-D) Validation of the screening results. SiRNA-mediated knockdown of JAK1 (C) and SMAD7 (D). Results are presented as ratios between the luciferase activity (± SD of Firefly/Renilla) obtained from cells transfected with specific siRNAs and cells transfected with scrambled siRNA. The knockdown efficiencies for JAK1 or SMAD7 also are shown together with their consequences for endogenous hepcidin mRNA expression. Levels of mRNA expression were normalized to the house-keeping gene GAPDH. Results are presented as fold change (± SD) compared with samples transfected with control siRNA. The mean of 3 independent experiments is shown. Significant changes are marked by *(P < .05) or **(P < .005).