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

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Figure 2. — SAFB phenocopies HOXA9 in leukemic cells. (A) Growth kinetics of MOLM13-Cas9 cells transduced with guide RNA (gRNA) (n = 3) targeting HOXA9 or SAFB. The data are shown as the average of biological replicates (n = 3) ± standard deviation (SD). Statistical significance was calculated against nontargeting control gRNA (nontreated [NT]) at time point days 4 and 5, using t test (2-tailed, P < .05), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (B) Western blot analyses showing the knockdown efficiency of HOXA9 and SAFB gRNAs in MOLM13 cells. β-tubulin is used as a loading control. (C) Apoptosis in MOLM13-Cas9 cells transduced with gRNA targeting HOXA9 (g5 and g7) or SAFB (g2 and g3) 5 days after transduction, as measured using annexin V and 7AAD staining. Plots are representative of 3 independent biological experiments. (D) Floating bar graphs summarizing results from the 3 independent experiments from apoptosis measurements using 3 gRNAs targeting HOXA9 (g5, g7, and g8) and SAFB (g1, g2, and g3) are shown. Statistical significance was calculated against NT using t test (2-tailed, P < .05), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. (E) Flow cytometric analyses of CD11b surface expression in MOLM13-Cas9 cells transduced with gRNA targeting HOXA9 (g5) or SAFB (g3). Contour plots shown here are representative of 3 independent biological replicates. (F) Floating bar graphs summarizing results from the 3 independent experiments from flow cytometric analyses of CD11b surface expression using 2 gRNAs targeting HOXA9 (g5 and g7) and SAFB (g2 and g3) are shown. Statistical significance was calculated against NT using t test (2-tailed, P < .05), ∗P < .05, ∗∗∗P < .001. (G) Western blot analyses showing expression of SAFB and HOXA9 in AML cell lines. β-Tubulin is used as a loading control. (H) CD11b surface expression in AML cell lines, 3 days after transduction, with gRNA targeting HOXA9 or SAFB (mean ± SD, n = 3). Statistical significance calculated using the 2-way analysis of variance (ANOVA) test, ∗∗P < .01. (I) Apoptosis measured via annexin V positivity in AML cell lines, 3 days after transduction, with gRNA targeting HOXA9 or SAFB (mean ± SD, n = 3). Statistical significance was calculated using the 2-way ANOVA test, ∗∗P < .01, ∗∗∗P < .001. (J) The mRNA expression of HOXA9 and SAFB in human AML primary samples (n = 179), AML The Cancer Genome Atlas (TCGA) data set. (K) Codependency between CRISPR knockout effects of HOXA9 and SAFB in AML cell lines (n = 26) from DepMap data set. Statistical significance was analyzed by linear regression at a 95% confidence interval (CI), P = .0016. The plot was generated using Prism. FITC, fluorescein isothiocyanate; KO, knockout.

Figure 2. — SAFB phenocopies HOXA9 in leukemic cells. (A) Growth kinetics of MOLM13-Cas9 cells transduced with guide RNA (gRNA) (n = 3) targeting HOXA9 or SAFB. The data are shown as the average of biological replicates (n = 3) ± standard deviation (SD). Statistical significance was calculated against nontargeting control gRNA (nontreated [NT]) at time point days 4 and 5, using t test (2-tailed, P < .05), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (B) Western blot analyses showing the knockdown efficiency of HOXA9 and SAFB gRNAs in MOLM13 cells. β-tubulin is used as a loading control. (C) Apoptosis in MOLM13-Cas9 cells transduced with gRNA targeting HOXA9 (g5 and g7) or SAFB (g2 and g3) 5 days after transduction, as measured using annexin V and 7AAD staining. Plots are representative of 3 independent biological experiments. (D) Floating bar graphs summarizing results from the 3 independent experiments from apoptosis measurements using 3 gRNAs targeting HOXA9 (g5, g7, and g8) and SAFB (g1, g2, and g3) are shown. Statistical significance was calculated against NT using t test (2-tailed, P < .05), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. (E) Flow cytometric analyses of CD11b surface expression in MOLM13-Cas9 cells transduced with gRNA targeting HOXA9 (g5) or SAFB (g3). Contour plots shown here are representative of 3 independent biological replicates. (F) Floating bar graphs summarizing results from the 3 independent experiments from flow cytometric analyses of CD11b surface expression using 2 gRNAs targeting HOXA9 (g5 and g7) and SAFB (g2 and g3) are shown. Statistical significance was calculated against NT using t test (2-tailed, P < .05), ∗P < .05, ∗∗∗P < .001. (G) Western blot analyses showing expression of SAFB and HOXA9 in AML cell lines. β-Tubulin is used as a loading control. (H) CD11b surface expression in AML cell lines, 3 days after transduction, with gRNA targeting HOXA9 or SAFB (mean ± SD, n = 3). Statistical significance calculated using the 2-way analysis of variance (ANOVA) test, ∗∗P < .01. (I) Apoptosis measured via annexin V positivity in AML cell lines, 3 days after transduction, with gRNA targeting HOXA9 or SAFB (mean ± SD, n = 3). Statistical significance was calculated using the 2-way ANOVA test, ∗∗P < .01, ∗∗∗P < .001. (J) The mRNA expression of HOXA9 and SAFB in human AML primary samples (n = 179), AML The Cancer Genome Atlas (TCGA) data set. (K) Codependency between CRISPR knockout effects of HOXA9 and SAFB in AML cell lines (n = 26) from DepMap data set. Statistical significance was analyzed by linear regression at a 95% confidence interval (CI), P = .0016. The plot was generated using Prism. FITC, fluorescein isothiocyanate; KO, knockout.