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. 2016 Jan 6;157(3):1146–1162. doi: 10.1210/en.2015-1747

Figure 4.

Figure 4.

Activin B (ACT B) induction of hepcidin depends on SMAD5, but not SMAD3 or SMAD2, in Hep3B cells. Hep3B cells were transfected with control siRNA (CTRL), siSMAD5, siSMAD3, or siSMAD2 (40nM) either alone (A) or in combination with the SMAD3-responsive firefly luciferase reporter (CAGA-Luc) and pRL-TK control Renilla luciferase vector (B and C). Forty-eight hours after transfection, cells were incubated in the absence or presence of 5-ng/mL ACT B, BMP6, or activin A (ACT A) as indicated for 6 hours, followed by measurement of HAMP relative to RPL19 mRNA levels by qRT-PCR (A) or relative luciferase activity (B and C). Data are expressed as the mean ± SEM from 3 separate experiments each performed in triplicate for the fold change relative to untreated control siRNA-transfected cells, which were normalized to 1. Statistical significance was determined by one-way ANOVA compared with control siRNA-transfected cells treated with the same ligand (*, P < .05; **, P < .01; ***, P < .001). For siSMAD5-transfected cells in panel A, fold change relative to untreated siSMAD5-transfected cells is indicated over the respective bars.