Hematopoietic progenitor cell colony forming assay from yolk sac and bone marrow of SIRT1+/+, SIRT1+/−, and SIRT1−/− mice. (A) Primitive erythroid colony formation from yolk sac (YS) cells generated from E8 to E8.25 SIRT1+/+, SIRT+/−, and SIRT1−/− embryos (SIRT1+/+, n = 11; heterozygous, n = 10; SIRT1−/−, n = 4) were grown in methylcellulose-based medium. Data are shown as mean ± SD; *P < .01. (B) PCR shows the genotyping of SIRT1 mutant embryos. The primers are: forward-5′-TCCTTGCCACAGTCACTCAC-3′, reverse for wild-type 5′-CATCTAAACTTTGTTGGCTGC-3′, reverse for deletion 5′-ACAGTCCCATTCCCATACC-3′. (C) Hematopoietic progenitor cell colony formation from bone marrow of 5-week-old SIRT1 heterozygote (SIRT1+/−) or wild-type controls (SIRT1+/+) under normoxia (20% O2) and 5% O2 conditions. Data are shown as mean ± SD; *P < .01; ND = SIRT1−/− cells were not available for use and were not done. (D) Hematopoietic progenitor cell colony formation from bone marrow of 12-month-old SIRT1 heterozygote (SIRT1+/−) or homozygote (SIRT1−/−) null mice or from wild-type controls (SIRT1+/+) under normoxia (20% O2) or 5% O2 conditions. (E) Cycling status of mouse bone marrow progenitor cells generated from 12-month-old SIRT1+/+, SIRT1+/−, and SIRT1−/− mice under normoxia (20% O2) and 5% O2 conditions. This shows percent progenitors in DNA synthesis (S-phase) as determined by high specific activity tritiated thymidine kill technique.23 P values were calculated with Student t test. ND = not done. Data are the average of 3-5 mice/group. (F) Survival of hematopoietic progenitor cells in vitro after 1 day delayed addition of cytokines as denoted by decreased colony formation. Results are expressed as mean ± ISEM for 4-5 mice per group.
