Hmgb3 expression correlates with long-term repopulating ability. (A) Separation of Lin^– c-kit^HI Hmgb3-KI bone marrow cells into GFP⁺ and GFP^– populations by flow cytometry (top). Sort gates were based on isotype controls (bottom). (B) Duplex RT-PCR performed on RNA isolated from sorted Hmgb3-KI bone marrow (BM) cells sorted into GFP⁺ and GFP^– populations. RT-PCR performed without RT (–RT) was used for a negative control. β2-microglobulin cDNA amplification was used as an internal control. The amplicon sizes are 154 bp and 258 bp for Hmgb3 and β2-microglobulin, respectively. (C) Mean CFU-GM frequencies in Lin^–, c-kit⁺, GFP^– (n = 3, 202 colonies counted), and GFP⁺ (n = 3, 69 colonies counted) populations. Colony-forming assays were performed as described in “Materials and methods.” P values were determined by Student t test. (D) Representative hemoglobin analysis of competitive repopulation of Lin^–, c-kit^HI, GFP⁺ versus GFP^– cells. Repopulations were performed with mixed doses of either 30 000 Lin^–, c-kit^HI, GFP⁺ (n = 5) or 50 000 Lin^–, c-kit^HI, GFP^– (n = 5) and 10⁷ C57BL/6 bone marrow as described in “Materials and methods.” 129/SvJ and C57BL/6 hemoglobin served as assay controls. The β^Min, β^Maj, (129/SvJ) and β^S (C57BL/6) hemoglobins are indicated by black arrows. The average erythroid repopulation by Hmgb3-KI bone marrow (determined as percentage of total Hb that is of 129/SvJ genetic origin) for each population is underneath their respective samples accompanied by the standard deviation. Hb levels were quantified by densitometry.