Skip to main content
. 2022 Apr 8;3(3):220–239. doi: 10.1158/2643-3230.BCD-21-0132

Figure 7.

Figure 7. Txnip preserves functional integrity of Dnmt3a-mutant HSCs via p21. A, Normalized level of BFP+ cell chimerism in donor-derived HSC (CD45.2+Lineage−c-Kit+EPCR+CD48−CD150+) pool 18 weeks posttransplant. Ratio for each mouse is normalized to average BFP+ HSC chimerism in PBS treatment for individual shRNAs for each genotype to account for differences in transduction efficiency (n = 4–5 per group). B, Number of BFP+ donor-derived HSCs (BFP+CD45.2+Lineage−c-Kit+EPCR+CD48−CD150+) in BM of recipients 18 weeks posttransplant. Data for two independent shRNAs targeting Txnip are compiled, denoted by square (Txnip-shRNA #1) or diamond (Txnip-shRNA #2) shapes. C, Schematic of functional genetic rescue by lentiviral shRNA transduction in H2B-GFP–labeled HSCs. D, Cleaved caspase 3/7 activity of shRNA-transduced HSCs (GFP+BFP+Lineage−c-Kit+EPCR+CD48−CD150+) purified from mice treated with PBS or IFNγ (two doses, cells purified 2 hours after the second dose). Apoptosis was quantified following a 16-hour culture period (n = 4–7). Data for two independent shRNAs targeting p53, p21 and Txnip are compiled. E, Time to first division of H2B-GFP–labeled HSCs (GFP+BFP+Lineage−c-Kit+EPCR+CD48−CD150+) of indicated genotypes transduced with Renilla-targeting shRNA (control) from mice treated with PBS or IFNγ (n = 4). F, Time to first division of H2B-GFP–labeled control or Dnmt3aKO HSCs (GFP+BFP+Lineage−c-Kit+EPCR+CD48−CD150+) transduced with shRNA targeting indicated genes from mice treated with IFNγ (n = 4). G, Schematic illustrating Txnip-p53-p21 axis in the preservation of Dnmt3a-mutant HSCs. One-way ANOVA (A), two-tailed t test (B, data are compared for IFNγ relative to PBS within each genotype/shRNA), two-way ANOVA (D and E) or one-way ANOVA with Tukey multiple test correction (F); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data represent mean ± SEM.

Txnip preserves functional integrity of Dnmt3a-mutant HSCs via p21. A, Normalized level of BFP+ cell chimerism in donor-derived HSC (CD45.2+Lineage−c-Kit+EPCR+CD48−CD150+) pool 18 weeks posttransplant. Ratio for each mouse is normalized to average BFP+ HSC chimerism in PBS treatment for individual shRNAs for each genotype to account for differences in transduction efficiency (n = 4–5 per group). B, Number of BFP+ donor-derived HSCs (BFP+CD45.2+Lineage−c-Kit+EPCR+CD48−CD150+) in BM of recipients 18 weeks posttransplant. Data for two independent shRNAs targeting Txnip are compiled, denoted by square (Txnip-shRNA #1) or diamond (Txnip-shRNA #2) shapes. C, Schematic of functional genetic rescue by lentiviral shRNA transduction in H2B-GFP–labeled HSCs. D, Cleaved caspase 3/7 activity of shRNA-transduced HSCs (GFP+BFP+Lineage−c-Kit+EPCR+CD48−CD150+) purified from mice treated with PBS or IFNγ (two doses, cells purified 2 hours after the second dose). Apoptosis was quantified following a 16-hour culture period (n = 4–7). Data for two independent shRNAs targeting p53, p21 and Txnip are compiled. E, Time to first division of H2B-GFP–labeled HSCs (GFP+BFP+Lineage−c-Kit+EPCR+CD48−CD150+) of indicated genotypes transduced with Renilla-targeting shRNA (control) from mice treated with PBS or IFNγ (n = 4). F, Time to first division of H2B-GFP–labeled control or Dnmt3aKO HSCs (GFP+BFP+Lineage−c-Kit+EPCR+CD48−CD150+) transduced with shRNA targeting indicated genes from mice treated with IFNγ (n = 4). G, Schematic illustrating Txnip-p53-p21 axis in the preservation of Dnmt3a-mutant HSCs. One-way ANOVA (A), two-tailed t test (B, data are compared for IFNγ relative to PBS within each genotype/shRNA), two-way ANOVA (D and E) or one-way ANOVA with Tukey multiple test correction (F); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data represent mean ± SEM.