Figure 7
Figure 7. Enforced coexpression of HOXB4 and sPrdm16 enhances HPC self-renewal in vitro and induces myeloid preleukemic transformation in transplanted mice. (A) Quantitative real-time PCR of endogenous Prdm16 mRNA levels in various sorted progenitor and stem cell populations. Gapdh mRNA was used as a loading control, and the level of Prdm16 mRNA expression in whole BM was set at a relative value of 1.0. (B) Schematic representation of lentiviral vectors to overexpress HOXB4 and sPrdm16. The promoters and fluorescent reporter genes used are indicated. The 400-bp chromatin insulator (i4r) was used as indicated to protect from position effects. (C) Western blot analysis of HOXB4 and sPrdm16 expression in NIH-3T3 cells transduced with the indicated vectors. Note the lower background band seen on the sPrmd16 blot is also present with the “empty” vector control. (D) Secondary colony numbers by myeloid CFU-C assay on sorted transduced cells as a measure of self-renewal capacity. Cells were transduced with control vectors or mock transduced or with single HOXB4 or sPrdm16 vectors or cotransduced with both HOXB4 and sPrdm16 vectors as indicated. After a primary CFU-C assay, cells were replated and colonies scored based on 10 000 cell inputs. Error bars show the standard deviation for multiple experiments, and statistical comparisons are indicated above the histograms. (E) Flow cytometry analyses of HOXB4 and sPrdm16 double-positive cells in peripheral blood (PB) and BMC of 2 primary recipients, # 890 and # 894. The top row shows staining for the donor background CD45.1, the middle row shows expression of the sPrdm16 vector marker (mCherry) and the HOXB4 vector marker (GFP), and the bottom row shows expression of the Mac-1 and Gr-1 myeloid markers. Note the expansion of double-positive myeloid cells in each of these cases. (F) BM cytospin photomicrographs of secondary recipients (#10037 and #10040) derived by transplantation from donor #890. Insets show higher magnification of the myeloid blasts seen in these cases, which comprised 30% to 50% of the cells on these slides. Slides were stained with Romanowsky stain, and images were acquired digitally on a Nikon ECLIPSE E800 microscope (Nikon, Japan) equipped with a Nikon DXM1200 camera and Nikon S Flour 40×/1.30 oil lens using NIS Elements software (Nikon). WT, wild-type.

Enforced coexpression of HOXB4 and sPrdm16 enhances HPC self-renewal in vitro and induces myeloid preleukemic transformation in transplanted mice. (A) Quantitative real-time PCR of endogenous Prdm16 mRNA levels in various sorted progenitor and stem cell populations. Gapdh mRNA was used as a loading control, and the level of Prdm16 mRNA expression in whole BM was set at a relative value of 1.0. (B) Schematic representation of lentiviral vectors to overexpress HOXB4 and sPrdm16. The promoters and fluorescent reporter genes used are indicated. The 400-bp chromatin insulator (i4r) was used as indicated to protect from position effects. (C) Western blot analysis of HOXB4 and sPrdm16 expression in NIH-3T3 cells transduced with the indicated vectors. Note the lower background band seen on the sPrmd16 blot is also present with the “empty” vector control. (D) Secondary colony numbers by myeloid CFU-C assay on sorted transduced cells as a measure of self-renewal capacity. Cells were transduced with control vectors or mock transduced or with single HOXB4 or sPrdm16 vectors or cotransduced with both HOXB4 and sPrdm16 vectors as indicated. After a primary CFU-C assay, cells were replated and colonies scored based on 10 000 cell inputs. Error bars show the standard deviation for multiple experiments, and statistical comparisons are indicated above the histograms. (E) Flow cytometry analyses of HOXB4 and sPrdm16 double-positive cells in peripheral blood (PB) and BMC of 2 primary recipients, # 890 and # 894. The top row shows staining for the donor background CD45.1, the middle row shows expression of the sPrdm16 vector marker (mCherry) and the HOXB4 vector marker (GFP), and the bottom row shows expression of the Mac-1 and Gr-1 myeloid markers. Note the expansion of double-positive myeloid cells in each of these cases. (F) BM cytospin photomicrographs of secondary recipients (#10037 and #10040) derived by transplantation from donor #890. Insets show higher magnification of the myeloid blasts seen in these cases, which comprised 30% to 50% of the cells on these slides. Slides were stained with Romanowsky stain, and images were acquired digitally on a Nikon ECLIPSE E800 microscope (Nikon, Japan) equipped with a Nikon DXM1200 camera and Nikon S Flour 40×/1.30 oil lens using NIS Elements software (Nikon). WT, wild-type.

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