GDF15 induction by iron depletion is independent of HIF. HeLa cells were seeded at 2 × 105 cells/mL and transfected at 24 hours and 48 hours with 200 nM HIF-1α siRNA, HIF-2α 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 mRNA and protein extraction. (A) qPCR quantification of GDF15 induction by BIP in the presence of various HIF siRNA treatments. (B) qPCR quantification of CA9 induction by BIP. (C) Western blotting for human HIF-1α and HIF-2α. Cell lysates were electrophoresed on SDS-polyaccrylamide gel, transferred onto cellulose acetate membrane, and stained with human monoclonal HIF-1α or HIF-2α antibodies. The housekeeping protein β-tubulin was stained as a loading control. (D,E) RCC4 cells stably transfected with empty vector or wild-type VHL-HA were seeded at 2 × 105 cells/mL and then cultured for 16 hours in the presence of 100 μM BIP. GDF15 and CA9 fold-induction by BIP compared with untreated cells was determined by qPCR after normalization to β-actin levels. In contrast with CA9, GDF15 was strongly induced by BIP irrespective of VHL status. *P < .05 compared with control siRNA with BIP.
