FIG. 3.

Progenitors having biphenotypic differentiation potential show strong expression of Hoxa9 and are clonal. (A) Identification of wild-type and mutant Hoxa9 proteins in immortalized myeloblasts from primary marrow. The abundance of Hoxa9 in total cell lysate was quantitated by immunoblot analysis using an antiserum raised against the HD C terminus of murine Hoxa9. The mobility of recombinant Hoxa9 equaled that of Hoxa9 expressed in myeloblast cell lines and is designated at the left as HoxA9. The higher mobility of the N-terminal EE-tagged Hoxa9 is designated at left as N-tag HoxA9. Lanes: 1, recombinant Hoxa9 produced by coupled transcription-translation; 2, M1AML cells containing coactivation of Hoxa9 and Meis1; 3 to 17, myeloid progenitors immortalized by Hoxb8 (lane 3), E2a-Pbx1 (lane 4); Hoxa9 (lanes 5 to 7), EE-tagged Hoxa9 (lanes 8 to 10), Hoxa9-WF (lanes 11 to 15), Hoxa9-Gtet (lane 16), and Hoxa9-Atet (lane 17). (B) Hoxa9-immortalized progenitors are clonal and thus exhibit biphenotypic potential. Shown is Southern blot analysis of DNA derived from Hoxa9-immortalized populations HF1 (lanes 1 and 5), HF1.1 (lanes 2 and 6), HF1.2 (lanes 3 and 7), and HF2 (lanes 4 and 8). DNA was cleaved with NheI and probed with Hoxa9 cDNA (lanes 1 to 4) or cleaved with BamHI and probed with neo sequences (lanes 5 to 8). The location of the genomic and proviral fragments containing Hoxa9 are indicated at the left. A background band present in all samples probed with neo sequences (lanes 5 to 8) is indicated at left as NS. The relative ratio of genomic to proviral Hoxa9 signal in lanes 1 to 3 of panel A was 1:1, confirming the presence of two proviral integrations, and was 2:1 in the sample of DNA in lane 4, confirming a single integration.