Transient Bcl-x_L induction contrasts with slower Bim suppression in response to Epo injection. (A) Flow cytometric histograms of Bim in freshly harvested splenic ProE. (Top panel) Histograms corresponding to single mice at the indicated time points after Epo injection, or 24 hours after control saline injection. Bim levels decrease (histograms shift to the left) after Epo injection. (Bottom panel) Bim levels in 6 saline-injected mice (6 histograms in various shades of blue) and 3 Epo-injected mice (histograms in shades of red/orange). The x-axis is in fluorescence units. (B) Bim protein levels, measured as illustrated in panel A, in adult erythroid differentiation subsets 3 days after a single injection of either Epo (red, 300 U/25 g) or saline (black). Data are mean ± SEM of n = 21 mice for saline injection, and n = 10 mice for Epo injection. *P < .00001 (2-tailed t test, unequal variance) for differences between Epo and saline-injected mice. (C) Time course of Bcl-x_L up-regulation (red symbols, plotted on the left, red-numbered y-axis) and Bim suppression (black symbols, plotted on the right, black-numbered y-axis) in spleen (circles) and BM (triangles) in response to a single Epo injection (300 U/25 g) on day 0. Includes a subset of the data plotted in panel B (for Bim) and Figure 2D (for Bcl-x_L). Bim data are mean ± SEM of n = 21 mice for day 0, and n = 3 to 10 mice for days 1 to 5, pooled from 5 experiments. Bim ProE curves (black lines) for spleen and BM were hand-drawn. Bim on day 0 was significantly different from subsequent days, where indicated: spleen ProE: *P < .005; spleen EryA: *P < .025; BM ProE: *P < .02; BM EryA: *P < .01 (2-tailed t test, unequal variance). (D) Time course of Bim_L and Bim_EL mRNA levels after a single Epo injection (300 U/25 g), in freshly isolated and sorted BM EryA and ProE. Each data point is the mean of triplicate qRT-PCR measurements on sorted cells from individual mice, expressed relative to the β-actin mRNA and normalized to t = 0 (saline-injected mice). There was a significant decrease after Epo injection in Bim_EL mRNA in ProE (P = .00439, 2-tailed t test, unequal variance, all post-Epo injection data were pooled) and EryA (P = .013), and in Bim_L mRNA in ProE (P = .027). (E) The ratio of Bim_L to Bim_EL mRNA in Epo-injected and in saline-injected mice, in the experiments described in panel D. Data are mean ± SD; all Epo time points were pooled for the purpose of this analysis. There were no significant changes in this ratio with Epo injection.