Fig. 3: EpoR regulates the speed of S phase.

a Cell-cycle analysis of Epor^−/− fetal liver cells transduced with either EpoR or Bcl-x_L and cultured as in Supplementary Fig. 2b. Cells were pulsed with BrdU for 30 min at t = 9 h and were immediately harvested for analysis. The fraction (%) of erythroblasts (hCD4⁺Lin⁻) in S phase is indicated, as is S phase speed, measured as the intra-S phase rate of BrdU incorporation (BrdU MFI within the S phase gate). b Summary of cell-cycle status and S phase speed, as measured by intra-S phase BrdU incorporation in EpoR or Bcl-x_L-transduced Epor^−/− fetal liver cells. Data is pooled from 6 independent experiments similar to (a). In all cases, cells were pulsed with BrdU for 30 min prior to harvesting for analysis. Data are mean ± sem. Intra-S phase BrdU (MFI) is expressed as the ratio to BrdU MFI of Bcl-x_L-transduced fetal liver cells at t = 0 in each experiment. Significance p-values are two-tailed paired t test, pairing EpoR and Bcl-x_L-transduced cells for each time point across all experiments (upper panel), and for t = 9 and t = 19 h in all experiments (middle and lower panels). c Effect of the cell-permeable iron carrier, Fe-SIH (10 μM) on S phase speed. Experiment and cell-cycle analysis as in (b). Cells were harvested at t = 9 h. d, e Summary of S phase speed (d) and cell-cycle status (e) in EpoR and Bcl-x_L-transduced Epor^−/− fetal liver cells at t = 9 h, experimental design as in Fig. 2b, and (a) to (c) above. S phase speed is expressed relative to the speed at t = 0 in each experiment. Shown are the effects of adding Fe-SIH or dN to the medium, or of doubly transducing cells with both Bcl-x_L and Tfrc. Data are mean ± sem for n = 4 independent experiments each for Fe-SIH and dN, and n = 3 for Tfrc. All experiments also had Epor^−/− fetal liver cells transduced with EpoR and with Bcl-x_L without additional additives or transductions. P-value is for a two-sided t test, unequal variance.