Hmgb3 mRNA levels in adult mouse tissues. (A) Northern blot analysis of Hmgb3 expression in adult mouse tissues. Top: Northern blot analysis of Hmgb3 expression in MEL, 32D cell lines, and CTLLs (left) and in adult mouse bone marrow (BM), spleen (Sp), thymus (Ty), brain (Br), heart (He), Liver (Li), Lung (Lu), and Kidney (Ki) (right). Signals corresponding to Hmgb3 mRNA and location of 18s and 28s rRNAs are indicated by arrows. The 3′ untranslated region was used as a probe (yellow bar, Figure 3, middle panel). Bottom: analysis of RNA loading and integrity. 15 μg RNA was loaded onto an agarose gel and stained with ethidium bromide following electrophoresis. (B) RT-PCR analysis of adult mouse bone marrow populations. RT-PCR analysis of bone marrow populations sorted by lineage: lineage-depleted cells, erythroid cells, platelets/megakaryocytes, granulocytes, monocytes/macrophages, mast cells, B cells, T cells, and whole bone marrow. Signals corresponding to Hmgb3 expression are indicated by black arrows. RT-PCR performed without RT (–RT) was used for a negative control. MEL RNA served as a positive control for Hmgb3 expression. β2-microglobulin (β2) was used for an internal control. (C) Semiquantitative duplex RT-PCR analysis of whole, Lin^–, c-kit⁺, Sca-1^–(L^–K^–S^–), and Lin^–, c-kit⁺, Sca-1⁺, IL-7Rα^– (L^–K⁺S⁺I^–) bone marrow cells. Hmgb3 mRNA levels were quantified within the linear range of amplification by densitometry and normalized to β2-microglobulin expression. Amplicon sizes are 359 bp and 258 bp for Hmgb3 and β2-microglobulin, respectively. (D) Hmgb3 mRNA in situ hybridization analysis. All analyses were performed on adult mouse lineage-depleted bone marrow cells sorted based on c-kit protein levels: c-kit^HI (upper left), c-kit^LO (middle left), and c-kit^NEG (lower left). Hybridization was performed with the same probe used for Northern analysis. All antisense hybridizations were performed in tandem with sense control hybridizations (upper, middle, and lower right), and the silver grains visualized by dark field microscopy. Examples of cells positive for Hmgb3 mRNA are indicated by arrows. Original magnification, × 400.