Decreased bcl-x_Lexpression in Stat5a^−/−5b^−/− early erythroblasts correlates with anemia.

Panels A-B show evaluation of a flow cytometry assay for bcl-x_L. (A) Histogram of HCD-57 cells labeled for bcl-x_L (x-axis: relative fluorescence). HCD-57 cells were cultured in Epo (1 U/mL) or starved of Epo for 18 hours in the presence of serum. Cells were then fixed, permeabilized, and labeled for bcl-x_L using a rabbit polyclonal antiserum (Pharmingen, see “Materials and methods,” here labeled as Pharm556361) and Alexa Fluor 488 goat anti–rabbit IgG. In the presence of Epo, 83% of cells label positive for bcl-x_L (marked by “M”); after 18 hours of starvation, this declines to 24%. (B) Bcl-x_Llevels in HCD-57 cells growing either in Epo (1 U/mL) or in SCF (100 ng/mL) assessed by flow cytometry or by Western blot analysis. Flow cytometry as described in panel A; 75% of cells are positive for bcl-x_L when cultured in Epo, but only 20% of cells express bcl-x_L when cultured in SCF. Western blot analysis was carried out either with a polyclonal rabbit IgG bcl-x_L(SC-634, Santa-Cruz Biotechnology, Santa Cruz, CA) or with the Pharm556361 antibody, as indicated. (C) A representative flow cytometry measurement of bcl-x_L in spleen erythroblasts. Spleen cells from one wild-type and one anemic Stat5a^−/−5b^−/− mouse were labeled with CD71 (APC channel) and Ter119 (PE channel). Cells were then fixed, permeabilized, and labeled for bcl-x_L (Alexa 488 channel). Bcl-x_L expression in CD71^high cells (which are >90% Ter119⁺ erythroblasts) is clearly reduced in Stat5a^−/−5b^−/− cells when compared with wild-type cells (right-hand panel). (D) Bcl-x_L expression in CD71^high cells was measured as described in panel C, in 5 wild-type mice and 6 Stat5a^−/−5b^−/− mice in the same experiment. Correlation line was fitted to the Stat5a^−/−5b^−/− data points (R² = 0.64). Representative of 3 similar experiments.