Stat5 Signaling Specifies Basal versus Stress Erythropoietic Responses through Distinct Binary and Graded Dynamic Modalities
Figure 5
High exogenous Stat5 levels rescue high-intensity graded p-Stat5 signaling in wild-type and EpoR-HM S3 cells.
(A) The p-Stat5 response to Epo in S3 cells expressing exogenous FLAG-Stat5. Wild-type fetal liver cells were electroporated with either FLAG-tagged Stat5a ( = FLAG-Stat5, C-terminal tag, top panels), FLAG-tagged STAT5Y694F (middle panels), or “empty vector” (pcDNA3, lower panels). Following overnight incubation in Epo (0.2 U/ml), cells were deprived of Epo for 3 h, and then either left unstimulated (left panels) or were stimulated with Epo for 30 min (response to Epo = 9 U/ml is shown; response to Epo concentrations 0.04 to 9 U/ml are shown in panels B, C). Double-headed arrows indicate persisting p-Stat5 following a 3h Epo deprivation, seen only in cells expressing high levels of FLAG-Stat5. (B) Analysis strategy for the experiment described in panel A. Dot plots of the p-Stat5 response versus FLAG-Stat5 levels are shown for three of the nine Epo concentrations examined (left panels; Epo = 0, 0.33, or 9 U/ml) in EpoR-HM S3 erythroblasts. Each dot plot was subdivided into narrow vertical gates, each containing cells of similar FLAG-Stat5 levels. Three of these gates, numbered 10 to 12, are highlighted in red, green, and blue, respectively. p-Stat5 responses to the three Epo concentrations in a given gate (left panels) were overlayed in a single histogram (right panels). For example, responses of cells in gate 10 (red) in each of the three left panels are all overlayed in the top right panel (framed red). (C) The “total p-Stat5” response to each of nine Epo concentrations for each vertical gate in panel B, fitted with a Hill curve. Analysis is shown for both wild-type (WT) S3 cells and for EpoR-HM S3 cells (HM). Gate numbers and Hill coefficients are indicated next to each curve. Data points are “total p-Stat5” (MFI±sem) for cells in a given gate. Each of the “total p-Stat5” MFI measurements was corrected by subtracting background fluorescence, given by the “total p-Stat5” MFI of cells expressing FLAG-Stat5Y694F in the same gate (see panel A). Gates 6 and 7 for wild-type cells and gates 9 and 10 for HM cells are re-plotted with an expanded y-axis. (D) Linear correlation between FLAG-Stat5 levels in each vertical gate, and the corresponding maximal p-Stat5 signal intensity (p-Stat5max), in wild-type cells (top panel, R2 = 0.96) and EpoR-HM S3 cells (lower panel, R2 = 0.998); analysis is of data shown in panel C. p-Stat5max is the maximal p-Stat5 response to Epo, defined by the Hill equation in Figure S3 and obtained by fitting Hill curves to the data in (C).