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. 2005 Mar 10;115(4):919–929. doi: 10.1172/JCI22725

Figure 1.

Figure 1

Immortalization of murine hematopoietic progenitors by MLL-SEPT6 fusion protein via aberrant expression of Hox genes. (A) Schematic representation of the retroviral constructions used. CXXC, CXXC domain; Zn fingers, zinc fingers; CS, cleavage sites; TAD, transactivation domain; SET, SET domain. (B) Western blot analysis of proteins extracted from PlatE cells transfected with the constructs shown in A, after immunoprecipitation using the anti-MLL Ab (lanes 1–9). Each lysate was blotted with the anti-FLAG Ab (lanes 1–9) or anti-SEPT6 Ab (lanes 10 and 11). Endogenous expression of SEPT6 was detected in lane 10. Lane 1, mock; lane 2, 5′-MLL; lane 3, MLL-SEPT6; lane 4, MLL-SEPT6Δcoil; lane 5, MLL-SEPT6Δcoil-ER; lane 6, MLL-SEPT6ΔGTP; lane 7, MLL-SEPT6ΔP-loop; lane 8, MLL-SEPT6S56N; lane 9, MLL-ENLs; lane 10, pMXs-neo alone (endogenous SEPT6); lane 11, SEPT6. (C) Experimental strategy for myeloid immortalization assay. (D) Myeloid immortalization assay using the constructs shown in A. Lanes for MLL-SEPT6Δcoil-ER indicate the presence (+) or absence (–) of 4-OHT. The bar graph shows numbers of colonies obtained after each round of replating in methylcellulose (average ± SD). (E and F) Typical morphology of the colonies generated by MLL-SEPT6 (E), and the cells constituting these colonies (F). Original magnification, ×40 (E), ×400 (F). (G) Expression of Hox a7, Hox a9, and Meis1 by RT-PCR in the cells from third-round cultures. β2m was used as an internal standard. M, 100-bp DNA ladder (New England Biolabs Inc.); lane 1, control (Ba/F3 with IL-3) cells; lane 2, mock; lane 3, MLL-SEPT6; lane 4, MLL-ENLs; lane 5, negative control.