Expression of erythroid and megakaryocytic markers in hematopoietic tissues from normal and phenylhydrazine (PHZ)-treated mice.
(A) Flow cytometric analysis of the expression of TER-119 (Y axes) and 4A5 (X axes) by bone marrow (top panels) and spleen (bottom panels) light density cells from normal and PHZ-treated (1, 2, and 3 days after the second injection) mice. The cells were also incubated with isotype-matched irrelevant antibodies as negative controls and the results presented on the left panels. R1, R2, and R3 indicate the gates used to define cells expressing TER-119 and 4A5 alone or coexpressing the 2 antigens. (The corresponding cell frequencies as determined in multiple experiments are presented in Table 2.) Propidium iodide–positive cells were less than 1% and were excluded by appropriate gating. (B) Hystogram analysis of the staining with FITC-IgG2 (right) or with FITC-4A5 (left) of normal (black line) and PHZ-treated (day 1, gray line) light density spleen cells gated in the TER-119–positive (R1+R3 of Figure 1A) area. (C) Semiquantitative RT-PCR analysis of the expression of erythroid (β-globin and EpoR), megakaryocytic (GpIIb,AchE, and Mpl) and myeloid (Mpo) genes in spleen cells obtained from either normal or PHZ-treated (days 1 and 3) mice. Actin was amplified to control for the amount of cDNA used in each reaction. β-globin and actin were amplified for 18, 21, and 24 cycles; Mpl for 25, 30, and 35 cycles; and all the other genes for 27, 30, and 33 cycles (increasing numbers of cycles are indicated by a triangle on the top of the panels). The results are representative of those obtained in 3 separate experiments.
