Figure 6
The effect of double expression of D171N and BMI1 in a mouse BMT model. (A) Kaplan-Meier analysis of the survival of mice that received transplants of BM cells transduced with pMYs.IG/BMI1 (n = 12, green line), D171N/pMYs.IN (n = 11, red line), or D171N/BMI1 (n = 12, blue line). P values were calculated using log-rank test. (B) Expression of RUNX1-D171N and BMI1 in BM cells derived from the BMT mice transduced with pMYs.IG/IN (lane 1), D171N/pMYs.IN (lanes 2, 3), or D171N/BMI1 (lanes 4-8). Cell lysates were immunoblotted with anti-Bmi1, anti–FLAG M2, or anti-tubulin antibody as control. Data are representative of 3 independent experiments. (C) Macroscopic findings of euthanized mice transplanted with BM cells transduced with the indicated construct. A representative photograph is shown. Mice with D171N/pMYs.IN or D171N/BMI1 died of MDS/AML with marked splenomegaly (right 2 panels), although mice with pMYs.IG/IN or pMYs.IG/BMI1 remained healthy without any organomegaly 8 months after BMT (left 2 panels). (D) Cytospin preparations of BM and spleen cells derived from indicated mice were stained with Giemsa. A representative photograph is shown. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); objective lens, UplanFl (Olympus); original magnification ×1000. (E) Flow cytometric analysis of BM cells derived from each transduced mouse. In pMYs.IG/IN and pMYs.IG/BMI1, apparently healthy mice were euthanized for analysis of BM cells 8 months after BMT. The dot plots show staining for NGFR, Gr-1, CD11b, B220, or c-kit as detected with PE vs GFP. (F) Histopathological findings of spleen and liver from mice that died of MDS/AML in the indicated BMT model, as shown by hematoxylin and eosin staining. Images were obtained with a BX51 microscope and a DP12 camera (Olympus) with an UplanFL objective lens (Olympus) and are shown at an original magnification ×400. (G) Ink4a/Arf (p16/p19) expression levels in BM cells of mice. Relative p16/p19 expression was measured by qRT-PCR performed in triplicate and calculated as the ratio of p16/p19 to Gapdh expression. (H) Evi1 expression levels in BM cells of mice. Relative Evi1 expression was measured by qRT-PCR performed in triplicate and calculated as the ratio of Evi1 to Gapdh expression. RNA from pMYs.IG/pMYs.IN mice served as a control, and the RNA level was defined as 1.

The effect of double expression of D171N and BMI1 in a mouse BMT model. (A) Kaplan-Meier analysis of the survival of mice that received transplants of BM cells transduced with pMYs.IG/BMI1 (n = 12, green line), D171N/pMYs.IN (n = 11, red line), or D171N/BMI1 (n = 12, blue line). P values were calculated using log-rank test. (B) Expression of RUNX1-D171N and BMI1 in BM cells derived from the BMT mice transduced with pMYs.IG/IN (lane 1), D171N/pMYs.IN (lanes 2, 3), or D171N/BMI1 (lanes 4-8). Cell lysates were immunoblotted with anti-Bmi1, anti–FLAG M2, or anti-tubulin antibody as control. Data are representative of 3 independent experiments. (C) Macroscopic findings of euthanized mice transplanted with BM cells transduced with the indicated construct. A representative photograph is shown. Mice with D171N/pMYs.IN or D171N/BMI1 died of MDS/AML with marked splenomegaly (right 2 panels), although mice with pMYs.IG/IN or pMYs.IG/BMI1 remained healthy without any organomegaly 8 months after BMT (left 2 panels). (D) Cytospin preparations of BM and spleen cells derived from indicated mice were stained with Giemsa. A representative photograph is shown. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); objective lens, UplanFl (Olympus); original magnification ×1000. (E) Flow cytometric analysis of BM cells derived from each transduced mouse. In pMYs.IG/IN and pMYs.IG/BMI1, apparently healthy mice were euthanized for analysis of BM cells 8 months after BMT. The dot plots show staining for NGFR, Gr-1, CD11b, B220, or c-kit as detected with PE vs GFP. (F) Histopathological findings of spleen and liver from mice that died of MDS/AML in the indicated BMT model, as shown by hematoxylin and eosin staining. Images were obtained with a BX51 microscope and a DP12 camera (Olympus) with an UplanFL objective lens (Olympus) and are shown at an original magnification ×400. (G) Ink4a/Arf (p16/p19) expression levels in BM cells of mice. Relative p16/p19 expression was measured by qRT-PCR performed in triplicate and calculated as the ratio of p16/p19 to Gapdh expression. (H) Evi1 expression levels in BM cells of mice. Relative Evi1 expression was measured by qRT-PCR performed in triplicate and calculated as the ratio of Evi1 to Gapdh expression. RNA from pMYs.IG/pMYs.IN mice served as a control, and the RNA level was defined as 1.

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