Figure 5.
Defects in RUNX1 methylation confer resistance to apoptosis induced by endogenous and genotoxic stress. (A) GSEA plots comparing Runx1+/+ and Runx1KTAMK/KTAMK LT-HSCs using the Gene Ontology biological process database. The top 3 gene sets enriched in control Runx1+/+ LT-HSCs are shown. Each solid bar represents one gene within the gene set. (B) Induction of ER stress–related genes by UPR in Runx1+/+ and Runx1KTAMK/KTAMK LT-HSCs as shown by quantitative polymerase chain reaction. Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) LT-HSCs were sorted and cultured with or without tunicamycin (0.6 μg/mL) for 10 hours (n = 7 from 4 mice per genotype; 6-8 weeks old, from 4 independent experiments). (C) Apoptosis by ER stress in Runx1+/+ and Runx1KTAMK/KTAMK LT-HSCs as detected by flow cytometry. Cells were treated with tunicamycin (0.6 μg/mL) for 20 hours. The percentages of Annexin V+ cells in Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) LT-HSCs are shown (n = 3 from 2 mice per genotype; 6 to 8 weeks old, from 2 independent experiments). (D) Induction of p53 downstream genes by radiation in Runx1+/+ and Runx1KTAMK/KTAMK Flt3– LSK cells as shown by quantitative polymerase chain reaction. Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) mice were irradiated (3 Gy), and Flt3– LSK cells were harvested 3 hours later (n = 4 to 5 mice; 8-15 weeks old, from 5 independent experiments). (E) Apoptosis by radiation in Runx1+/+ and Runx1KTAMK/KTAMK Flt3– LSK cells as detected by flow cytometry. Mice were irradiated (3 Gy), and Flt3– LSK cells were harvested 18 hours later. The left panel shows a representative flow cytometry plot of Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) Flt3– LSK cells with (solid lines) or without (dashed lines) radiation. The percentages of Annexin V+ cells in Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) cells are shown in the right graph (n = 3 mice; 9-14 weeks old, from 3 independent experiments). (F) Flow cytometry analysis of Runx1+/+, Runx1+/KTAMK, and Runx1KTAMK/KTAMK Flt3– LSK cells for γH2AX staining. Mice were irradiated (3 Gy) and Flt3– LSK cells were harvested 18 hours later. The left panel shows a representative flow cytometry plot of Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) Flt3– LSK cells with (solid lines) or without (dashed lines) radiation. The fluorescence minus one (FMO) control is shown in orange. The percentages of γH2AX+ cells in Runx1+/+ (blue), Runx1+/KTAMK (light green), and Runx1KTAMK/KTAMK (red) cells are shown in the right graph (n = 3 mice; 7-10 weeks old, from 3 independent experiments). FDR, false discovery rate; NES, normalized enrichment score; NS, not significant.

Defects in RUNX1 methylation confer resistance to apoptosis induced by endogenous and genotoxic stress. (A) GSEA plots comparing Runx1+/+ and Runx1KTAMK/KTAMK LT-HSCs using the Gene Ontology biological process database. The top 3 gene sets enriched in control Runx1+/+ LT-HSCs are shown. Each solid bar represents one gene within the gene set. (B) Induction of ER stress–related genes by UPR in Runx1+/+ and Runx1KTAMK/KTAMK LT-HSCs as shown by quantitative polymerase chain reaction. Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) LT-HSCs were sorted and cultured with or without tunicamycin (0.6 μg/mL) for 10 hours (n = 7 from 4 mice per genotype; 6-8 weeks old, from 4 independent experiments). (C) Apoptosis by ER stress in Runx1+/+ and Runx1KTAMK/KTAMK LT-HSCs as detected by flow cytometry. Cells were treated with tunicamycin (0.6 μg/mL) for 20 hours. The percentages of Annexin V+ cells in Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) LT-HSCs are shown (n = 3 from 2 mice per genotype; 6 to 8 weeks old, from 2 independent experiments). (D) Induction of p53 downstream genes by radiation in Runx1+/+ and Runx1KTAMK/KTAMK Flt3– LSK cells as shown by quantitative polymerase chain reaction. Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) mice were irradiated (3 Gy), and Flt3– LSK cells were harvested 3 hours later (n = 4 to 5 mice; 8-15 weeks old, from 5 independent experiments). (E) Apoptosis by radiation in Runx1+/+ and Runx1KTAMK/KTAMK Flt3– LSK cells as detected by flow cytometry. Mice were irradiated (3 Gy), and Flt3– LSK cells were harvested 18 hours later. The left panel shows a representative flow cytometry plot of Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) Flt3– LSK cells with (solid lines) or without (dashed lines) radiation. The percentages of Annexin V+ cells in Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) cells are shown in the right graph (n = 3 mice; 9-14 weeks old, from 3 independent experiments). (F) Flow cytometry analysis of Runx1+/+, Runx1+/KTAMK, and Runx1KTAMK/KTAMK Flt3– LSK cells for γH2AX staining. Mice were irradiated (3 Gy) and Flt3– LSK cells were harvested 18 hours later. The left panel shows a representative flow cytometry plot of Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) Flt3– LSK cells with (solid lines) or without (dashed lines) radiation. The fluorescence minus one (FMO) control is shown in orange. The percentages of γH2AX+ cells in Runx1+/+ (blue), Runx1+/KTAMK (light green), and Runx1KTAMK/KTAMK (red) cells are shown in the right graph (n = 3 mice; 7-10 weeks old, from 3 independent experiments). FDR, false discovery rate; NES, normalized enrichment score; NS, not significant.

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