Figure 6
Figure 6. In vitro and in vivo requirement of E2F3 for BCR/ABL leukemogenesis. (A) IL3-dependent and -independent methylcellulose colony forming ability of primary Lin− E2F3+/+ and E2F3−/− C56BL/6J BMCs retrovirally transduced with either the empty MigR1 or MiGR1-p210BCR/ABL retrovirus and GFP sorted (P < .001; t test). Error bars represent standard error. (B) Nested RT-PCR for p210-BCR/ABL in peripheral blood isolated at 12 weeks after transplantation from SCID mice (nos. 1 and 2) injected with E2F3+/+ and E2F3−/− Lin− BMCs. Sensitivity of the system was assessed by detecting p210-BCR/ABL transcripts in K562/32Dcl3 cells used at 1:106 ratio. (C) Visual analysis of spleens isolated from controls, E2F3+/+, or E2F3−/− BCR/ABL-transduced GFP+-BCR/ABL+Lin− SCID mice. (D) Hematoxylin/eosin-stained BM, spleen, and liver tissue sections from control, E2F3+/+ or E2F3−/− BCR/ABL-transduced GFP+-BCR/ABL+Lin− SCID mice (nos. 1 and 2) killed at 15 weeks after transplantation. (E) Representative nested RT-PCR for p210-BCR/ABL in peripheral blood isolated at 3 weeks after transplantation from SCID mice either not injected (control; n = 3) or injected with vector-transduced (wild-type; n = 3) or shRNA E2F3ab–transduced (shE2F3; n = 12) 32D-BCR/ABL cells. (F) Visual analysis and average weight of spleens isolated from controls, wild-type, and shE2F3 mice at 3.5 weeks after transplantation. Error bar represent standard error. (G) Representative hematoxylin/eosin staining of BM and spleen tissue sections from control, wild-type, and shE2F3 mice at 3.5 weeks after transplantation. (H) Survival of SCID mice (n = 13 per group) intravenously injected with vector-transduced (blue line) and E2F3ab shRNA-transduced (red line) 32D-BCR/ABL cells. Estimated probabilities for survival were calculated using the Kaplan-Meier method, and the log-rank test evaluated the differences among survival distributions (P < .001). Images were taken with a Zeiss (Thornwood, NY) Axioskope 2 Plus and a 40×/0.75 (BM) or a 25×/0.75 (liver and spleen) NA objective, with a Canon Powershot A70 (Canon, Lake Success, NY) and Canon Capture software.

In vitro and in vivo requirement of E2F3 for BCR/ABL leukemogenesis. (A) IL3-dependent and -independent methylcellulose colony forming ability of primary Lin− E2F3+/+ and E2F3−/− C56BL/6J BMCs retrovirally transduced with either the empty MigR1 or MiGR1-p210BCR/ABL retrovirus and GFP sorted (P < .001; t test). Error bars represent standard error. (B) Nested RT-PCR for p210-BCR/ABL in peripheral blood isolated at 12 weeks after transplantation from SCID mice (nos. 1 and 2) injected with E2F3+/+ and E2F3−/− Lin− BMCs. Sensitivity of the system was assessed by detecting p210-BCR/ABL transcripts in K562/32Dcl3 cells used at 1:106 ratio. (C) Visual analysis of spleens isolated from controls, E2F3+/+, or E2F3−/− BCR/ABL-transduced GFP+-BCR/ABL+Lin− SCID mice. (D) Hematoxylin/eosin-stained BM, spleen, and liver tissue sections from control, E2F3+/+ or E2F3−/− BCR/ABL-transduced GFP+-BCR/ABL+Lin− SCID mice (nos. 1 and 2) killed at 15 weeks after transplantation. (E) Representative nested RT-PCR for p210-BCR/ABL in peripheral blood isolated at 3 weeks after transplantation from SCID mice either not injected (control; n = 3) or injected with vector-transduced (wild-type; n = 3) or shRNA E2F3ab–transduced (shE2F3; n = 12) 32D-BCR/ABL cells. (F) Visual analysis and average weight of spleens isolated from controls, wild-type, and shE2F3 mice at 3.5 weeks after transplantation. Error bar represent standard error. (G) Representative hematoxylin/eosin staining of BM and spleen tissue sections from control, wild-type, and shE2F3 mice at 3.5 weeks after transplantation. (H) Survival of SCID mice (n = 13 per group) intravenously injected with vector-transduced (blue line) and E2F3ab shRNA-transduced (red line) 32D-BCR/ABL cells. Estimated probabilities for survival were calculated using the Kaplan-Meier method, and the log-rank test evaluated the differences among survival distributions (P < .001). Images were taken with a Zeiss (Thornwood, NY) Axioskope 2 Plus and a 40×/0.75 (BM) or a 25×/0.75 (liver and spleen) NA objective, with a Canon Powershot A70 (Canon, Lake Success, NY) and Canon Capture software.

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