Stat5 Signaling Specifies Basal versus Stress Erythropoietic Responses through Distinct Binary and Graded Dynamic Modalities
Figure 3
Binary p-Stat5 signaling in EpoR-HM mice and in mature wild-type S3.
(A) Representation of the cytoplasmic domains of wild-type EpoR or its truncated mutants, EpoR-H and EpoR-HM. Tyrosine residues are represented by red lines. Tyrosine 343 is the only remaining tyrosine in EpoR-H, and is mutated in EpoR-HM. (B) The p-Stat5 response to Epo (2 U/ml, 30 min) in S1 cells from wild-type (WT), EpoR-H (H), or EpoR-HM (HM) fetal livers on E13.5. Percentage of cells in the p-Stat5+ gate is indicated. (C) Fluorescence p-Stat5 histogram overlay of S1 cells from E13.5 wild-type (top) or EpoR-HM (bottom), stimulated with a range of Epo concentrations for 30 min. Representative of three similar experiments. S1 cells in each case are pooled from several fetal livers of the same genotype. (D) Epo dose/p-Stat5 response curves, in wild-type or EpoR-HM S1 cells; the p-Stat5 MFI data correspond to the histograms shown in (C). Data are fitted with Hill curves. The Hill curves that were fitted to the data in the top panel were re-plotted in the bottom panel and are shown as a fraction of the maximal p-Stat5 response (p-Stat5max). The p-Stat5max was calculated from fitting the Hill equation to the experimental “total p-Stat5” data. Hill coefficients (nH) are indicated. (E) Lower panel, table summarizing the Hill coefficients (nH) obtained by fitting the Hill curve to plots of “total p-Stat5 versus Epo concentration,” for each of subsets S1 to S3, in each of three independent experiments. Fetal liver cells were pooled from three or four embryos of each genotype in each experiment. Differences between nH for EpoR-HM and WT are significant at p = 0.01, paired t test. R2 is Pearson's product moment correlation coefficient, correlating experimental data with values predicted by the Hill equation for the corresponding Epo concentrations. (F) CD71/Ter119 profile of E14.5 fetal livers (left panel). The S3 subset was further divided into serial FSC gates, each corresponding to 300 channels (middle panel). The right panel shows an overlay of Ter119 expression in FSC gates #3 and #6. (G) Epo dose/p-Stat5 response curves for the S3 FSC gates #3–6 and for S1 cells in the same fetal liver. Gate number and corresponding Hill coefficient are shown for each curve.