GDF15 mRNA and protein are induced by iron chelation in human cell lines. Cells were seeded at 2 × 105 cells/mL and then cultured for 16 hours alone (untreated), in the presence of 100 μM BIP or at 0% oxygen (anoxia). Cells were then harvested for mRNA extraction and cDNA synthesis. Supernatants were collected and stored at −20°C for use in ELISA. (A) qPCR quantification of GDF15 expression. GDF15 signal was normalized to β-actin signal in the respective sample, and fold change in GDF15 calculated relative to untreated cells. (B) ELISA measurement of secreted GDF15 protein in cell supernatants. Serial dilutions of the recombinant human GDF15 were used as standards. (C) qPCR quantification of CA9 expression. CA9 signal was normalized to β-actin signal in the respective sample, and fold change in expression calculated relative to untreated cells. (D) Cells were seeded at 2 × 105 cells/mL and then cultured for 16 hours alone (untreated) with 100 μM BIP, or at 0% oxygen (anoxia), or in the presence of both BIP and anoxia (combined). Cells were then harvested for mRNA extraction and cDNA synthesis, GDF15 signal was normalized to β-actin signal in the respective sample, and fold change in GDF15 calculated relative to untreated cells. *P < .05 compared with untreated cells. NS indicates not significant.
