Figure 4
Figure 4. Fasudil is effective against transplanted AML cells in vivo. (A) Schematic outline of the treatment regime. After 10 weeks, animals were either fasudil treated (n = 12, 50 μg/g/d) or control treated (n = 13). After 2 weeks of treatment, mice were euthanized, and the percentage of human cells in the murine BM was assessed by flow cytometry. (B) Comparison of the percentage of human chimerism (percentage of human CD45-positive per total CD45-positive cells in the BM) between fasudil-treated (red) and control-treated animals (black). The difference between fasudil-treated and control-treated mice was statistically significant by Mann-Whitney U test (P = .036). (C) Overview of the experiments to address the effect of shRNA-mediated ROCK1 knockdown on the growth of primary cells from AML2 in NSG mice. At the end of the experiment, murine BM from each mouse was analyzed by flow cytometry, and human cells identified by positivity for human CD45 and human CD33 were flow sorted to purity. The sorted cells were spilt and grown in the presence or absence of puromycin to assess the transduction rate. (D) The human chimerism in murine bone marrow 12 weeks after transplant with AML2 cells containing nontarget control shRNAs (scramble, n = 8) or shRNAs targeting ROCK1 (n = 10). Compared with mice receiving scramble-transduced cells, those animals receiving ROCK1 shRNA cells had a significantly lower median chimerism (0.6% vs 1.6%; P = .011 by Mann-Whitney U test). Note that the chimerism in both groups is lower than in the experiments shown in panel B, probably due to the prolonged ex vivo culture period needed for transduction. (E) Comparison of the transduction rate in ROCK1 shRNA–transduced and scramble-transduced cells before (start) and 12 weeks after (end) transplant into NSG mice. Although the transduction rate at both time points was comparable in cells transduced with nontarget control shRNAs, it decreased to almost 0 in ROCK1 shRNA–transduced cells (median, 33.8% vs 1.85%; P < .001). In panels B and D, horizontal lines depict the median within each group.

Fasudil is effective against transplanted AML cells in vivo. (A) Schematic outline of the treatment regime. After 10 weeks, animals were either fasudil treated (n = 12, 50 μg/g/d) or control treated (n = 13). After 2 weeks of treatment, mice were euthanized, and the percentage of human cells in the murine BM was assessed by flow cytometry. (B) Comparison of the percentage of human chimerism (percentage of human CD45-positive per total CD45-positive cells in the BM) between fasudil-treated (red) and control-treated animals (black). The difference between fasudil-treated and control-treated mice was statistically significant by Mann-Whitney U test (P = .036). (C) Overview of the experiments to address the effect of shRNA-mediated ROCK1 knockdown on the growth of primary cells from AML2 in NSG mice. At the end of the experiment, murine BM from each mouse was analyzed by flow cytometry, and human cells identified by positivity for human CD45 and human CD33 were flow sorted to purity. The sorted cells were spilt and grown in the presence or absence of puromycin to assess the transduction rate. (D) The human chimerism in murine bone marrow 12 weeks after transplant with AML2 cells containing nontarget control shRNAs (scramble, n = 8) or shRNAs targeting ROCK1 (n = 10). Compared with mice receiving scramble-transduced cells, those animals receiving ROCK1 shRNA cells had a significantly lower median chimerism (0.6% vs 1.6%; P = .011 by Mann-Whitney U test). Note that the chimerism in both groups is lower than in the experiments shown in panel B, probably due to the prolonged ex vivo culture period needed for transduction. (E) Comparison of the transduction rate in ROCK1 shRNA–transduced and scramble-transduced cells before (start) and 12 weeks after (end) transplant into NSG mice. Although the transduction rate at both time points was comparable in cells transduced with nontarget control shRNAs, it decreased to almost 0 in ROCK1 shRNA–transduced cells (median, 33.8% vs 1.85%; P < .001). In panels B and D, horizontal lines depict the median within each group.

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