![Figure 2. Vav-Atmin∆/∆ LT-HSCs have impaired functions during regeneration and aging. Experiments were performed with young (A-H and Q-T) and aged (I-P) BM cells. Black bars/symbols and lines represent Atminf/f, and open symbols/gray bars and lines represent Vav-Atmin∆/∆, unless otherwise indicated. (A,I) Cell cycle distribution was determined using Ki67/DAPI stains in BM LT-HSCs (n = 4/genotype). (B, J) qRT-PCR for the expression of Cdkn1c and Rbl2 in LT-HSCs. Expression values are relative to the mean of control (n = 3-5/genotype). Other cell cycle regulators were unchanged between ATMIN-deficient and control cells (data not shown). (C,K) To estimate the stem/progenitor cell capacity of the more primitive cells, the frequency of L−S+K+ cells to form CAFCs was determined by limiting dilution 5 weeks after seeding on MS5 stroma. (D,L) Five-week LT culture-initiating cell–derived colonies were also determined (n = 3/genotype). (E,M) To evaluate the regeneration capacity of LT-HSCs, peripheral blood (PB) chimerism was determined at different time points from sublethally irradiated NSG ([nonobese diabetic/severe combined immunodeficiency disease/interleukin-2 receptor γ-chain–null] mice; CD45.1 due to the mixed background) that were transplanted with 15 LT-HSCs from either (E) young or (M) aged Atminf/f or Vav-Atmin∆/∆ mice (CD45.2). After 16 weeks, 15 LT-HSCs were sorted by flow cytometry from primary mice and retransplanted into separate secondary recipients, and PB chimerism was determined at different time points. (F,N) Percentage of engrafted LT-HSCs 16 hours after IV injection of total Atminf/f or Vav-Atmin∆/∆ BM mononuclear cells (n = 2 independent experiments). Each symbol indicates a mouse, and horizontal lines represent median reconstitution levels. (G,O) qRT-PCR for the expression of the indicated DNA oxidation repair genes in LT-HSCs was also evaluated. Expression values were relative to the mean of each control subpopulation (n = 3/genotype). (H, P) pKap1 expression in LT-HSCs was determined by intracellular flow cytometry analysis. Gray and open histograms represent isotype-matched control and pKap1 stains, respectively (n = 3/genotype). (Q) Hematopoietic reconstitution was monitored by serial PB counts in mice injected with a single dose of 5-FU. Total white blood cell (WBC) counts are shown (n = 5-6/genotype). Cell cycle status (R) and pKap1 expression (S) in ATMIN-deficient and control LT-HSCs at day 7 after a single dose of 5-FU administration (n = 4-5/genotype). (T) Kaplan-Meier survival curve of Atminf/f and Vav-Atmin∆/∆ mice injected 3 times (arrows) with 5-FU (n = 7-8/genotype). Unless stated, mean and standard deviation values are shown. *P < .03; **P < .003; ***P < .0003.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/128/16/10.1182_blood-2015-09-672980/4/2017f2.jpeg?Expires=1735824596&Signature=tdCHe9P~g1YI0l-IvEZD4XAkZcKFqi7kNI05H9cj4S4pm26I2Kuac430aDdKPw3237oiM2-dVPB9LCivGqpvxVg0PLk1y8V9q3UphJflMNfSW~qksIE7r2c1DU0-hpRbTt7Oz2lJDwnuIS0gM4uS3MAfALTncAfvfGPdqZO~cYxe-Lcbn2E~sW9o8xrdDU-wn2~iYYc2IrddC58vGiK2klyZkyXj7RMh5ess5fsYsZXiWx-qgX5Q4cFaqNpne2ehDgZIqEdQFa9do8xXcr5t5eeFQADoIMOVzc1K4LGO6h3GTD~IMnprbxAo0M83zA4t7ZofcygsrOYL4Sbvj2g7Qg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Vav-Atmin∆/∆ LT-HSCs have impaired functions during regeneration and aging. Experiments were performed with young (A-H and Q-T) and aged (I-P) BM cells. Black bars/symbols and lines represent Atminf/f, and open symbols/gray bars and lines represent Vav-Atmin∆/∆, unless otherwise indicated. (A,I) Cell cycle distribution was determined using Ki67/DAPI stains in BM LT-HSCs (n = 4/genotype). (B, J) qRT-PCR for the expression of Cdkn1c and Rbl2 in LT-HSCs. Expression values are relative to the mean of control (n = 3-5/genotype). Other cell cycle regulators were unchanged between ATMIN-deficient and control cells (data not shown). (C,K) To estimate the stem/progenitor cell capacity of the more primitive cells, the frequency of L−S+K+ cells to form CAFCs was determined by limiting dilution 5 weeks after seeding on MS5 stroma. (D,L) Five-week LT culture-initiating cell–derived colonies were also determined (n = 3/genotype). (E,M) To evaluate the regeneration capacity of LT-HSCs, peripheral blood (PB) chimerism was determined at different time points from sublethally irradiated NSG ([nonobese diabetic/severe combined immunodeficiency disease/interleukin-2 receptor γ-chain–null] mice; CD45.1 due to the mixed background) that were transplanted with 15 LT-HSCs from either (E) young or (M) aged Atminf/f or Vav-Atmin∆/∆ mice (CD45.2). After 16 weeks, 15 LT-HSCs were sorted by flow cytometry from primary mice and retransplanted into separate secondary recipients, and PB chimerism was determined at different time points. (F,N) Percentage of engrafted LT-HSCs 16 hours after IV injection of total Atminf/f or Vav-Atmin∆/∆ BM mononuclear cells (n = 2 independent experiments). Each symbol indicates a mouse, and horizontal lines represent median reconstitution levels. (G,O) qRT-PCR for the expression of the indicated DNA oxidation repair genes in LT-HSCs was also evaluated. Expression values were relative to the mean of each control subpopulation (n = 3/genotype). (H, P) pKap1 expression in LT-HSCs was determined by intracellular flow cytometry analysis. Gray and open histograms represent isotype-matched control and pKap1 stains, respectively (n = 3/genotype). (Q) Hematopoietic reconstitution was monitored by serial PB counts in mice injected with a single dose of 5-FU. Total white blood cell (WBC) counts are shown (n = 5-6/genotype). Cell cycle status (R) and pKap1 expression (S) in ATMIN-deficient and control LT-HSCs at day 7 after a single dose of 5-FU administration (n = 4-5/genotype). (T) Kaplan-Meier survival curve of Atminf/f and Vav-Atmin∆/∆ mice injected 3 times (arrows) with 5-FU (n = 7-8/genotype). Unless stated, mean and standard deviation values are shown. *P < .03; **P < .003; ***P < .0003.
