Figure 3. Loss of Alox5 impairs the function of CML stem cell.

a, BCR-ABL-expressing (GFP⁺) and non-BCR-ABL-expressing (GFP) Lin⁻c-Kit⁺Sca-1⁺ cells (CML stem cells) in BM and the spleens (SPL) were analyzed by FACS in recipients of BCR-ABL-transduced BM cells from wild type or Alox5^−/− donor mice (n=4 for each group at two time points after BMT). Total number of GFP⁺Lin⁻c-Kit⁺Sca-1⁺ cells for each mouse was calculated as percentage of GFP⁺Lin⁻c-Kit⁺Sca-1⁺ cells x total cell count for the cells from femurs and tabias. Loss of Alox5 caused significant reduction of CML stem cells in BM (p< 0.05). b, Equal numbers of the sorted GFP⁺Lin⁻c-Kit⁺Sca-1⁺ cells from recipients of BCR-ABL-transduced bone marrow cells (5000 cells each) from wild type (CD45.1) or Alox5^−/−(CD45.2) donor mice were mixed, followed by transplantation into lethally irradiated wild type mice. At days 14 and 25 after BMT, FACS analysis showed that the percentages of CD45.1⁺ cells were much higher than those of CD45.2⁺ cells. All these mice died of CML, presumably due to the development of CML from CD45.1⁺ cells. c, GFP⁺Lin⁻c-kit⁺Sca-1⁺ cells sorted by FACS from BCR-ABL-transduced BM cells from wild type or Alox5^−/− mice were injected into lethally irradiated wild type recipient mice (15000 GFP⁺Lin⁻c-kit⁺Sca-1⁺ cells per recipient mouse). At day 14 after BMT, GFP⁺ Gr-1⁺ cells in peripheral blood of the mice were analyzed by FACS. Alox5-deficient GFP⁺Lin⁻c-kit⁺Sca-1⁺ poorly engrafted. The mice receiving the Alox5-deficient GFP⁺Lin⁻c-kit⁺Sca-1⁺ cells survived (n=4), whereas the mice receiving the wild type GFP⁺Lin⁻c-kit⁺Sca-1⁺ cells died of CML (n=2). d, FACS analysis indicated the percentages of BCR-ABL-expressing (GFP⁺) and non-BCR-ABL-expressing (GFP) CMP, GMP, and MEP cells in BM of recipients of BCR-ABL-transduced wild type or Alox5^−/− donor BM cells (n=4). The results showed that loss of Alox5 caused depletion of BCR-ABL-expressing but not non-BCR-ABL-expressing CMP, GMP, and MEP cells in BM of the mice.