Iron deficiency, hypoxia, and iron overload control soluble HJV production through the regulation of furin. (A) Transfected HeLa cells were incubated for 8 hours in serum-free media in the presence of 300 μM DFO. Total lysates and concentrated media (50 μg) were analyzed using anti-HJV, anti-furin, and anti–HIF-1α. α-Tubulin was used to verify equal loading. The histogram indicates the FUR promoter activity, expressed as a fold induction of the luciferase reporter gene normalized on the empty vector. The Renilla activity was used to verify the transfection efficiency. Experiments were replicated 3 times. (B, top panel) Scheme of the experiment shown in the bottom panel. (B, bottom panel) Cells were treated with DFO, to activate HIF-1α, in the presence (+) or absence (−) of 50 μM CMK, to inhibit furin. Total lysates and concentrated media (50 μg) were analyzed by Western blot, using anti-HJV. The relative molecular mass, in kilodaltons, is indicated on the left. (C,D) Transfected cells were treated with 500 μM CoCl₂ (C) or 100 μM FAC (D) for 24 hours and analyzed as indicated in panel A. U indicates untreated; DFO, deferoxamine; CoCl₂, cobalt chloride; FAC, ferric ammonium citrate; s-HJV, soluble HJV; c-HJV, cell-associated HJV; and CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone. Error bars indicate SD. *P < .01. **P < .005. ***P < .001.