GDF15 expression in erythroblasts requires TfR2, GDF15 production levels were measured in the supernatants of cultured cells. The data shown corresponds to the production of GDF-15 over 24 hours for 10⁶ cells. (A) Determination of GDF15 levels secreted by erythroid progenitors on each day of the cell culture after CD36⁺ cell sorting. The results of 1 representative experiment from 3 independent cultures are shown. (B) Cells at day 9 of differentiation were incubated for 24 hours in the absence of Epo. The culture medium was then recovered and GDF15 concentration was determined by ELISA. (C) Determination of GDF15 secretion in control cells (Control Sh) and TfR2 knockdown cells (expressing shRNAs A or B). The data shown are the means ± SD determined on day 9 of 2 independent erythroid primary cultures (CD36⁺ cells). (D) Relationship between Epo concentrations in the culture medium and GDF15 production in UT7 cells. Cells were incubated for 24 hours with the indicated concentrations of Epo. After that time, GDF15 production was determined using the cell culture supernatants and intracellular ATP content of the cells was determined as described in “Methods.” (E) Determination of GDF15 secretion in UT7 control cells and in TfR2 knockdown cells expressing shRNAs A and B. Cells were cultured in the presence of 2 U/mL Epo. The data shown are the means ± SD from 4 independent experiments. **P < .01 relative to control.