GDF15 induction by iron depletion is independent of IRP. HeLa cells were seeded at 2 × 105 cells/mL and transfected at 24 hours and 48 hours with 200 nM IRP1 siRNA, IRP2 siRNA, or a combination of both. dHIF siRNA was used as an irrelevant control siRNA. After the second transfection, cells were treated with 100 μM BIP for a further 16 hours, after which cells were harvested for RNA extraction. (A) qPCR quantification of GDF15 induction by BIP in the presence of various IRP siRNA treatments. (B) qPCR quantification of transferrin receptor 1 (TfR1) induction by BIP. TfR1 was used as a control for an IRP1- and IRP2-dependent iron-responsive gene. (C) qPCR quantification of IRP1 expression in cells treated with IRP1 siRNA alone, IRP2 siRNA alone, or with a combination of siRNAs. (D) qPCR quantification of IRP2 expression in cells treated with IRP1 siRNA alone, IRP2 siRNA alone, or with a combination of siRNAs. *P < .05 compared with control siRNA without BIP. **P < .05 compared with control siRNA with BIP.
