Reducing Id2 levels rescues impaired erythroid development of Gfi-1 knockout BM in vitro and in vivo. (A) Schematic representation of the BFU-E colony assay. A total of 5 × 10⁴ BMC or 7.5 × 10⁴ spleen cells were cultured in MethoCult to enumerate BFU-E for 7 to 10 days. Colonies were stained with benzidine as described in “Methods.” (B) Representative photomicrographs (original magnification ×40) of BFU-Es from BMCs and large CFU-mix (1.5-2 mm) from stem progenitor cells. Benzidine-stained colonies were identifiable by the dark brown/black color of the colonies. Grid on culture dish is 2 mm × 2 mm. (C) Bar graph showing total number of the stained BFU-E colonies per plate, which can be directly compared because BM cellularity is equivalent between mice. Large colonies showing typical BFU-E shape that were first identified as red hemoglobinized colonies and then confirmed by benzidine staining were enumerated. These data were obtained by counting triplicate samples and are representative of 3 independent experiments. The data are presented as the mean ± SD. *P < .05 for Gfi-1^−/− compared with Gfi-1^−/−;Id2^+/− BFU-E. (D) Bar graph showing total number of CFU-S₈ counted in the spleens of recipient mice. The majority of CFU-S₈ are erythroid-committed early progenitors. The data were obtained by counting triplicate spleens and are representative of 2 separate experiments. The data are presented as mean ± SD. *P < .05 for Gfi-1^+/+ compared with Gfi-1^−/−, and Gfi-1^−/− compared with Gfi-1^−/−;Id2^+/− CFU-S₈. (E) Schematic presentation of transplantation experiment (top). RBC counts in the peripheral blood of individual recipients measured 15, 22, 29, and 36 days after BMT (bottom). Column scatterplot showing RBC numbers in recipient mice (n = 10 per group). Data are representative of 2 separate experiments. SP, stem progenitor.