Changes in idEpoRic Localization and STAT5 Phosphorylation After CID Stimulation in Fetal Liver Hematopoietic Cells.

<p>(A) Flowcytometry of the fetal liver cells from wild-type (WT) or line A (Tg) mouse embryos at E12.5. Ter119 and c-Kit expressions levels were analyzed by GFP (idEpoRic) expression, and the percentages of cells in each quadrangle are shown (blue letters in dot plot data). The GFP⁺ fraction was analyzed using an anti-Ter119 antibody (green) or an isotype control antibody (as a negative control [NC], black line) in the top histogram. Cells positive for Ter119 or c-Kit have been analyzed by GFP expression, and black-line histograms show the data from wild-type littermates (middle and bottom histograms). The percentages of cells in the gated regions are shown in each panel. (B) The subcellular localization of idEpoRic in the E12.5 fetal liver was examined using laser confocal microscopy. Single cell suspensions from wild-type (WT) and transgenic (Tg) fetal livers at E12.5 were incubated with (bottom) or without (top and middle) 1 nmol/mL CID for 15 minutes, and the cells were observed after nuclear staining with Hoechst33342 (blue). Note the membrane-associated localization of GFP (green) in the transgenic cells (middle) and the internalization of the GFP⁺ clusters after CID stimulation (arrowheads). A GFP-negative cell (asterisk) is found in a preparation of the transgenic cells. The scale bar indicates 10 μm. (C) Intracellular phosphorylated STAT5 (p-STAT5) in E12.5 fetal liver cells was detected using PE-conjugated anti-phosphorylated STAT5(Y694)-specific antibodies. Cells from line A fetal liver were exposed to rHuEPO (2.0 U/mL) or CID (1.0 nmol/mL) for 0 (Pre), 15, or 180 minutes. The left panels show basal levels of p-STAT5 in the transgenic (black lines) and wild-type (WT, blue) cells compared with the isotype antibody controls (NC, gray).