Figure 3.

Increased apoptosis and proliferation without overt senescence of bystander hematopoietic cells. (A) Apoptosis of the resided hematopoietic cells. Apoptosis was measured in homed Lin⁻Sca-1⁺ cells with multicolor flow cytometry combining surface markers of CD45.1, CD45.2, lineage, Sca-1 with annexin V, and DAPI. Plots were gated on homed donor Lin⁻Sca-1⁺ cells (left). Percentage of early apoptosis (annexin V⁺DAPI⁻) and late apoptosis was compared between NR and IR recipients (right). *P < .05. n = 3 or 4. (B) Cell proliferative potential. Transplanted BMNCs were labeled with CFSE before injection. Three days later, the division of donor Lin⁻ cells in NR or IR recipients was analyzed according to the intensity of CFSE in flow cytometry. The representative plots were generated from ModFit Version 3.1 software. (C) Senescence of the bystander cells from the irradiated hosts. Homed Lin⁻Sca-1⁺ cells from NR and IR were sorted and stained with X-gal for detecting the β-gal activity. Blue color in the cells indicated positive activity of β-gal. Lin⁻Sca-1⁺ cells cultured in the medium containing 50 ng/mL of stem cell factor, 50 ng/mL of Flt3, and 10 ng/mL of thrombopoietin at 37°C, 5% CO₂ for 10 days were used as positive control. Real-time RT-PCR was performed for examining the mRNA expression of p16^INK4A and p19^ARF in homed cells 17 hours after transplantation. M indicates DNA ladder. Lanes 1 and 2 represent homed Lin⁻Sca-1⁺ cells from IR recipients; lanes 3 and 4, homed Lin⁻Sca-1⁺ cells from NR recipients; and lanes 5 and 6, positive controls.