The contribution of TRIM28 to erythropoiesis is erythroid cell autonomous. (A) Experimental design. Total bone marrow donor cells isolated from Trim28^flox/flox:Tg^Mx1Cre (flox/flox:Mx1Cre) or control Trim28^flox/flox (flox/flox) mice (CD45.2) were coinjected with an approximately equal number of wild-type competitive donor cells (CD45.1) into lethally irradiated wild-type recipient mice (CD45.1). (B) CD45.2 and CD45.1 expression in each population in peripheral blood (before Cre induction) or in bone marrow (2 weeks post Cre activation) were analyzed by flow cytometry (left panel). Peripheral blood cells were analyzed 6 weeks after transplantation for CD45.2⁺ donor cell contribution in the Mac1⁺Gr1⁺ myeloid (PB_M) and B220⁺CD19⁺ B lymphoid (PB_B) populations. Total bone marrow cells were analyzed 2 weeks after completing poly(I:C) treatment for CD45.2⁺ donor cell contribution to the HSC/MPP, CMP, MEP, and GMP compartments. Each circle represents the average of control (black circle) or TMC mutant cells (open circle) with SEM (left panel). Data represent the summary of 8 recipient mice of each genotype from 2 independent experiments. Because erythroblasts and later-stage erythroid cells are negative for CD45,³⁵  the contribution of CD45.2⁺ (male) cells to the immature erythroid cell fraction was analyzed by qPCR, quantifying the abundance of Sry genomic DNA (Y chromosome; right panel). The average of Trim28^flox/flox mice was set to 1. Male CD45.2 and female CD45.1 cells were used in the first experiment. Each circle represents an individual mouse, and black bars represent the average (right panel). The summary of the first experiment with 4 recipients of each donor genotype is shown. In the second set of experiments, female CD45.2 and male CD45.1 cells were used; an (statistically insignificant) increased contribution of coinjected competitive CD45.1 cells was observed (data not shown). (C) The absolute number of different stages of erythroid cells in the bone marrow (2 femurs and 2 tibias). Mixtures of CD45.2 and CD45.1 cells are depicted. Average with SEM. * indicates statistically significant, P < .05. NS: not significant, P > .05.