Gdf15 signaling activates Hif2α-dependent Bmp4 expression via inhibition of Vhl during the recovery from anemia. (A-B) Analysis of relative mRNA expression levels of Bmp4 in the spleen at key time points during the recovery from BMT (A) and PHZ challenge (B), respectively. Each time point reflects >3 mice analyzed. Data are shown as individual subject and the mean ± SEM. (C) Primary spleen cells isolated from Gdf15^−/− mice were treated ±10 ng/mL GDF15 for the indicated time. Relative Bmp4 and Vhl mRNA expression levels were determined by qRT-PCR. Each time point reflects >3 mice analyzed. Data are shown as individual subject and the mean ± SEM. (D) Primary spleen cells derived from Gdf15^−/− mice were transduced with short hairpin RNAs targeting Vhl gene for 48 hours and then cultured in stress erythropoiesis media for 72 hours. Relative mRNA expression levels of Bmp4 and Vhl were analyzed by qRT-PCR. Each treatment was done in triplicate and the histogram is representative of 2 independent experiments. Data are shown as the mean ± SEM. (E-F) Analysis of relative mRNA expression levels of Vhl in the spleen at key time points during the recovery from (E) BMT and (F) PHZ challenge, respectively. Each time point reflects >3 mice analyzed. Data are shown as individual subject and the mean ± SEM. (G-H) Chromatin immunoprecipitation analysis of Hif2α binding to HRE4 of Bmp4 gene in the spleen during the recovery from BMT (G) and PHZ (H) treatment. Binding affinity is expressed as fraction of input DNA. Each time point reflects >3 mice analyzed. Data are shown as the mean ± SEM. (I-J) Analysis of Vhl and Hif2α protein expression in the spleen during the recovery from BMT (I) and PHZ (J) challenge, respectively. Western blots were probed with anti-VHL, anti-HIF2α, and anti-β-actin (control) antibodies. Three independent experiments were performed. In quantification graphs, data from 1 independent experiment are shown as individual subject and the mean ± SEM. *P < .05, **P < .01, and ***P < .001.