Figure 3.
ASXL1-MT and HHEX cooperatively promoted myeloid transformation in RUNX1-ETO and FLT3-ITD. (A) RUNX1-ETO–expressing human CB cells were transduced with vector/ASXL1-MT (coexpressing GFP) and vector/HHEX (coexpressing NGFR). Shown are the changes of GFP/NGFR double-positive (DP) cell frequency from 2 independent experiments (left) and the relative proliferation rate at the end of culture (right, n = 4). (B) The frequency of S/G2/M-phase cells in each DP fraction of the cells described in panel A was normalized to that in the double-negative fraction at day 7 (n = 4). (C) The frequency of Annexin V–positive cells in each DP fraction of the cells described in panel A at day 7 (n = 4). (D) Representative cytospin preparation of RUNX1-ETO–expressing cells transduced with indicated constructs on day 30 (left; scale bars, 20 μm), and the distribution of mature, immature, and blast-like cells (right; n = 3). (E) Schematic presentation of RUNX1-ETO9a–induced leukemia model. (F) Kaplan-Meier curve for the survival of the transplanted mice in each group (Control+EV, n = 15; ASXL1-MT-KI+EV, n = 17; Control+HHEX, n = 15; ASXL1-MT-KI+HHEX, n = 17; log-rank test). (G) Representative cytospin preparation of BM and spleen cells of RUNX1-ETO9a/ASXL1-MT/HHEX induced leukemic mice (scale bars, 20 μm). (H) Composition of the indicated surface markers in RUNX1-ETO9a induced leukemia cells infiltrating BM (Control+EV, n = 3; ASXL1-MT-KI+EV, n = 3; Control+HHEX, n = 4; ASXL1-MT-KI+HHEX, n = 6). (I) Kaplan-Meier curve for the survival of the transplanted mice in each group (Control+EV, n = 10; ASXL1-MT-KI+EV, n = 11; Control+HHEX, n = 11; ASXL1-MT-KI+HHEX, n = 11; log-rank test). (J) Composition of the indicated surface markers in FLT3-ITD induced leukemia cells infiltrating BM (Control+EV, n = 3; ASXL1-MT-KI+EV, n = 3; Control+HHEX, n = 5; ASXL1-MT-KI+HHEX, n = 5). Statistical analyses were performed by 1-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.

ASXL1-MT and HHEX cooperatively promoted myeloid transformation in RUNX1-ETO and FLT3-ITD. (A) RUNX1-ETO–expressing human CB cells were transduced with vector/ASXL1-MT (coexpressing GFP) and vector/HHEX (coexpressing NGFR). Shown are the changes of GFP/NGFR double-positive (DP) cell frequency from 2 independent experiments (left) and the relative proliferation rate at the end of culture (right, n = 4). (B) The frequency of S/G2/M-phase cells in each DP fraction of the cells described in panel A was normalized to that in the double-negative fraction at day 7 (n = 4). (C) The frequency of Annexin V–positive cells in each DP fraction of the cells described in panel A at day 7 (n = 4). (D) Representative cytospin preparation of RUNX1-ETO–expressing cells transduced with indicated constructs on day 30 (left; scale bars, 20 μm), and the distribution of mature, immature, and blast-like cells (right; n = 3). (E) Schematic presentation of RUNX1-ETO9a–induced leukemia model. (F) Kaplan-Meier curve for the survival of the transplanted mice in each group (Control+EV, n = 15; ASXL1-MT-KI+EV, n = 17; Control+HHEX, n = 15; ASXL1-MT-KI+HHEX, n = 17; log-rank test). (G) Representative cytospin preparation of BM and spleen cells of RUNX1-ETO9a/ASXL1-MT/HHEX induced leukemic mice (scale bars, 20 μm). (H) Composition of the indicated surface markers in RUNX1-ETO9a induced leukemia cells infiltrating BM (Control+EV, n = 3; ASXL1-MT-KI+EV, n = 3; Control+HHEX, n = 4; ASXL1-MT-KI+HHEX, n = 6). (I) Kaplan-Meier curve for the survival of the transplanted mice in each group (Control+EV, n = 10; ASXL1-MT-KI+EV, n = 11; Control+HHEX, n = 11; ASXL1-MT-KI+HHEX, n = 11; log-rank test). (J) Composition of the indicated surface markers in FLT3-ITD induced leukemia cells infiltrating BM (Control+EV, n = 3; ASXL1-MT-KI+EV, n = 3; Control+HHEX, n = 5; ASXL1-MT-KI+HHEX, n = 5). Statistical analyses were performed by 1-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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