Bcl-xL is induced in adult early erythroblasts in response to Epo injection. (A) Time course of plasma Epo, assayed by ELISA, after a single subcutaneous injection of 300 U/25 g body weight. Two mice (identified as either circles or triangles) were assayed per time point. (B) Gating strategy for freshly explanted adult splenic erythroid subsets ProE, EryA, EryB, and EryC.5 Live cells were selected, and subsets gated based on Ter119, CD71, and forward scatter (FSC). The spleen illustrated in this panel was harvested 18 hours after a subcutaneous Epo injection. All axes units refer to fluorescence. (C) Representative flow cytometric histograms of Bcl-xL in the indicated spleen erythroblast subsets. Anti–Bcl-xL antiserum was used to stain erythroblasts from a saline-injected mouse (blue histograms), or an Epo-injected mouse (300 U/25 g, red histograms) in freshly explanted spleen at 18 hours postinjection. Nonimmune serum (IgG) was used to measure the nonspecific binding in each subset (gray histograms). The x-axis is in fluorescence units. (D) Bcl-xL expression measured as in panel C in freshly explanted spleen, in each erythroblast subset at each of the indicated time points after a single Epo injection (300 U/25 g). Each data point for Epo-injected mice is mean ± SEM of n = 4 mice for t = 16, 18, 24, 48 hours, and mean of 2 mice for t = 12 hours. Blue curves are mean ± SEM of n = 14 saline-injected mice pooled from all time points. The same blue curves are reproduced for comparison with Epo-injected mice at each time point. Statistical significance values: ProE at t = 16 hours, in BM *P = .005, in spleen *P = .0006. EryA in spleen, *P = .0009 at 16 hours, *P = .0009 at 18 hours. EryA in BM, *P = .013 at 16 hours, *P = .015 at 18 hours. The induction of Bcl-xL in splenic EryA was significantly higher than in BM EryA (*P = .021). Two-tailed t test with unequal variance was used for all comparisons. (E) Epo dose/Bcl-xL response in vivo in spleen EryA. Wild-type Balb/C mice were injected subcutaneously with either saline (= basal, blue circle) or a single dose of Epo (1, 3, 10, 20, 30, or 300 U/25 g, red circles). Bcl-xL was measured by flow cytometry as in panel C at 18 hours postinjection, with the nonspecific fluorescence reading subtracted for each subset. Data from 2 independent experiments were pooled and normalized. Data points were fitted with a Hill curve. Each data point represents mean ± SEM of n = 3 to 4 mice. (F) Time course of Bcl-xL mRNA levels after a single Epo injection (300 U/25 g), in freshly isolated and sorted spleen and BM EryA. Quantitative real-time PCR (qRT-PCR), data points are mean ± SEM of 3 independent experiments. Data are expressed relative to the β-actin mRNA and normalized to the value in BM EryA in saline-injected mice.
