EKLF is required for PlGF expression, and iron overload is significantly related to mortality. Enforced EKLF expression in EKLF knockout erythroid cells increases (A) hemoglobin and (B) PlGF expression, whereas EKLF knockout erythroid cells are not able to respond in the same manner, suggesting that EKLF is required for PlGF induction. (C-D) Quantitative PCR results from anti-EKLF ChIP. EK-1 cells were treated as described in “Materials and methods.” Immunoprecipitation was carried out with anti-EKLF. After immunoprecipitation, quantitative PCR was performed to determine the amount of DNA precipitated. (E) HO-1 and (F) PlGF expression in K562 with indicated chemicals. ST638 was incubated with K562 for 30 minutes before hemin was included. (G) Hemin-induced PlGF expression was attenuated by NAC treatment. K562 cells were treated with indicated concentrations of NAC 30 minutes before hemin induction. Cells were harvested after 24 hours, RNA was extracted, and quantitative PCR was performed on cDNA preparations. (H) Patients with HH the most common form of iron overload disease, at the time of initial diagnosis have elevated levels of serum ferritin and PlGF (r = 0.241, P = .06, Spearman correlation). When ferritin levels are lowered by phlebotomy therapy, the PlGF concentration significantly decreases (P = .0013).