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. 2022 Dec 30;141(14):1737–1754. doi: 10.1182/blood.2022016528

Figure 3.

Figure 3.

HOXA9 and SAFB drive leukemic growth in vivo. (A) Cumulative growth of MOLM13 cells, lentivirally expressing shRNA against HOXA9 (blue), SAFB (red), scrambled sequence (orange), and empty vector (EV, gray). The data shown are the averages of biological replicates (n = 3) ± SD. Two-way ANOVA test, ∗P < .01 (B) Western blot analyses showing the knockdown efficiency of SAFB shRNAs after 48 hours after induction with doxycycline (Dox) in MOLM13 cells (top). β-Tubulin was used as a loading control. The HOXA9 knockdown after 7 and 10 days of doxycycline induction in MOLM13 cells is shown (bottom). (C) Myeloid differentiation accessed by CD11b surface expression via flow cytometry 5 days after induction with doxycycline. Data shown are the averages of biological replicates (n = 4) ± SD. Statistical significance calculated using the 2-way ANOVA test, ∗P < .01. (D) Percentage of annexin V– and 7AAD-positive MOLM13 cells 5 days after doxycycline treatment (1.5 μg/mL). Mean ± SD, n = 4. Two-way ANOVA test, ∗∗P < .001. (E) Colony-forming assay of MOLM13 cells expressing HOXA9- or SAFB-shRNA in the presence or absence of doxycycline (1.5 μg/mL). The bar graph shows the average value of 3 independent experiments ± SD, 2-way ANOVA test ∗∗P < .001, ∗∗∗P = .0001. (F) Schematic of xenotransplant experimental design. (G) Bioluminescent radiance 3 days after injection and before shRNA induction (baseline) in all 3 cohorts in all animals. (H) Serial bioluminescence imaging of mice that underwent transplantation with luciferase-labeled shRNA-expressing MOLM13 cells at indicated time points. (I) Bioluminescence at indicated time points shows disease progression over time. Statistical significance was calculated using the 2-way ANOVA with multiple comparisons (95% CI) against shEV, (shEV-HOXA9-sh at day 11, ∗∗∗P = .0004; shEV-SAFB-sh at day 11, ∗∗∗∗P < .0001). (J) Kaplan-Meier plot showing the survival of mice that received transplantations with MOLM13 cells expressing indicated shRNA. A log rank test was performed (∗∗P < .01, ∗∗∗P < .001). H9, HOXA9sh; Max, maximum; Min, minimum; Scr, scrambled.