GDF15 is induced by intracellular iron depletion. HeLa cells were seeded at 2 × 105 cells/mL 8 hours before transfection. Cells were transfected with 1 μg/well PQCXIX mammalian expression vector encoding wild-type ferroportin (WT FPN), inactive mutant V162Δ ferroportin (V162Δ FPN), or an irrelevant insert fused to EGFP (irrelevant vector). (A) Cells were harvested at 24, 48, and 72 hours after transfection and GDF15 mRNA were determined by qPCR. *P < .05 compared with irrelevant vector. (B) ELISA measurement of secreted GDF15 protein in cell supernatants collected 48 hours after transfection. *P < .05 compared with irrelevant vector. (C,D) HeLa cells were transfected with 1 μg/well PQCXIX mammalian expression vector encoding β2M-HFE (WT), inactive mutant β2M-HFE V100Δ (mut HFE), or FuGene transfection reagent alone (mock transfection). Cells were harvested 72 hours after transfection and GDF15 mRNA and CA9 mRNA determined by qPCR. For 36 hours before harvest, cells were treated with or without 2 mg/mL holotransferrin. *P < .05 compared with mock transfection. Plotted data are mean plus or minus SD of 3 independent biologic replicates.
