Figure 6
Figure 6. FRATN blocks Rac activation and inhibits leukemia induction by MLL-ENL. (A) Western blot analysis of Rac1 and GTP and total Rac1 in preleukemic and leukemic wild-type B6ME (B6) and F123ME (F123) cells, transduced with control and FRATN-expressing retroviral vectors. Rac2 GTP and total Rac2 are also shown in leukemic B6ME cells. Numbers represent densitometric quantitation of Rac GTP levels normalized to total Rac protein bands. α-tubulin served as a loading control. Data were obtained from individual preleukemic and leukemic cell lines in each case and are representative of 3 independent experiments. *The leukemic B6ME Rac1 blot shown is a shorter exposure compared with the other lanes. (B) Number of colonies formed in methylcellulose by purified hCD2T+ leukemic wild-type MLL-ENL cells transduced with control (Con) and FRATN-expressing (FRATN) retroviral vectors. Bars represent means of duplicate values; and error bars, the SD. **P < .01 versus control. Data are representative of 4 independent experiments. (C) Plots represent apoptosis in leukemic wild-type MLL-ENL cells 5 days after transduction with control and FRATN-expressing retroviral vectors. Bar chart represents the mean percentages of early-stage (annexin V+PI−) and late-stage (annexin V+PI+) apoptotic cells in triplicate cultures; and error bars, the SD. Data are representative of 4 independent experiments. (D) Kaplan-Meier survival curves for mice transplanted with wild-type MLL-ENL leukemic cells, after transduction with control and FRATN-expressing retroviral vectors. Group numbers are shown. These data are representative of 2 independent experiments. (E) Western blot analysis of Rac1 and Rac2 GTP and total Rac in human THP1 cells transduced with control (−) and FRATN-expressing (+) lentiviral vectors (left panel). Numbers represent densitometric quantitation of Rac GTP levels normalized to total Rac protein bands. α-tubulin served as a loading control. Graph represents relative cell number-transduced THP1 cells as measured by MTS assay. Bars represent mean values obtained from 3 independently transduced cultures; and error bars, the SD. ***P < .001 versus control. (A-D) At 48 hours after transduction, hCD2T+ transduced cells were positively selected by MACS and either used directly for the experiments (A-B,D) or equivalent numbers plated into culture and analyzed 3 days later (C).

FRATN blocks Rac activation and inhibits leukemia induction by MLL-ENL. (A) Western blot analysis of Rac1 and GTP and total Rac1 in preleukemic and leukemic wild-type B6ME (B6) and F123ME (F123) cells, transduced with control and FRATN-expressing retroviral vectors. Rac2 GTP and total Rac2 are also shown in leukemic B6ME cells. Numbers represent densitometric quantitation of Rac GTP levels normalized to total Rac protein bands. α-tubulin served as a loading control. Data were obtained from individual preleukemic and leukemic cell lines in each case and are representative of 3 independent experiments. *The leukemic B6ME Rac1 blot shown is a shorter exposure compared with the other lanes. (B) Number of colonies formed in methylcellulose by purified hCD2T+ leukemic wild-type MLL-ENL cells transduced with control (Con) and FRATN-expressing (FRATN) retroviral vectors. Bars represent means of duplicate values; and error bars, the SD. **P < .01 versus control. Data are representative of 4 independent experiments. (C) Plots represent apoptosis in leukemic wild-type MLL-ENL cells 5 days after transduction with control and FRATN-expressing retroviral vectors. Bar chart represents the mean percentages of early-stage (annexin V+PI−) and late-stage (annexin V+PI+) apoptotic cells in triplicate cultures; and error bars, the SD. Data are representative of 4 independent experiments. (D) Kaplan-Meier survival curves for mice transplanted with wild-type MLL-ENL leukemic cells, after transduction with control and FRATN-expressing retroviral vectors. Group numbers are shown. These data are representative of 2 independent experiments. (E) Western blot analysis of Rac1 and Rac2 GTP and total Rac in human THP1 cells transduced with control (−) and FRATN-expressing (+) lentiviral vectors (left panel). Numbers represent densitometric quantitation of Rac GTP levels normalized to total Rac protein bands. α-tubulin served as a loading control. Graph represents relative cell number-transduced THP1 cells as measured by MTS assay. Bars represent mean values obtained from 3 independently transduced cultures; and error bars, the SD. ***P < .001 versus control. (A-D) At 48 hours after transduction, hCD2T+ transduced cells were positively selected by MACS and either used directly for the experiments (A-B,D) or equivalent numbers plated into culture and analyzed 3 days later (C).

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