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Blood, Vol. 111, Issue 3, 1700-1708, February 1, 2008
Adherence to macrophages in erythroblastic islands enhances erythroblast proliferation and increases erythrocyte production by a different mechanism than erythropoietin
Blood Rhodes et al.
111: 1700
Supplemental materials for: Rhodes et al
Files in this Data Supplement:
- Figure S1. Analysis of cell cycle in FVA erythroblasts (JPG, 264 KB)
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Adherent and control FVA erythroblast populations are not synchronized for phase of cell cycle. In order to study a cohort of cells in the same phase of cell cycle, the erythroblasts were pulsed for 30 min with BrdU at 6 h, washed in fresh unlabeled medium, and recultured in fresh unlabeled medium in which the label is “chased” until 15 h of total culture (8 h of chase time). Aliquots of the cells were examined by flow cytometry at 7 h and at 2 h intervals thereafter by dual fluorescence: with BrdU fluorescence to detect the cells that were in S phase at the time of the pulse and with propidium iodide fluorescence to determine phase of cell cycle by total DNA content at the time the aliquot was taken. The pulsing, washing, and reculturing at 6 h required approximately one hour. Thus, the 7 h time represented the “pulsed” erythroblasts without any chase, and the 7 h erythroblasts that have BrdU fluorescence the green dots in the histograms are all in S phase because the incorporation of BrdU label during the pulse period is restricted to cells in DNA synthesis. As expected, the total DNA distribution of pulse-labeled 7 h erythroblasts extends between 2N and 4N. However, because of the Gaussian nature of these distributions, the total DNA distribution of some erythroblasts in the earliest stages of S-phase is actually slightly overlapped with the Gaussian distribution of 2N erythroblasts in G0/G1 phase seen in later samples of the chase period. Estimates of overlap between G0/G1-phase and S-phase: By 9 hrs (2 hrs of chase), erythroblasts have begun to undergo mitosis and show a distinct G0/G1 population (having only 2N DNA) which we used as a template to construct a G0/G1 box designated as R5 in all histograms in Figure S1 The two sets of BrdU-labeled cells, those in late S phase + G2/M and those in G0/G1, were sufficiently separated in total DNA content at 9 h that the R5 box demarcating the location of the G0/G1 cells in both adherent and non-adherent erythroblast histograms could be easily drawn. In Figure S1, these G0/G1 populations at 9 h were 20.57% of BrdU-labeled cells in adherent erythroblasts and 22.83 % of BrdU–labeled cells in control cells. This same R5 box was then applied to all histograms. In the 7 h pulsed samples in which 100% of BrdU-labeled erythroblasts were in S phase, 12.86% of adherent and 13.08% of control erythroblasts overlapped the R5 (G0/G1) box. Thus, at the beginning of the chase period, both adherent and control cells were approximately equal in terms of percentage of S-phase overlap in the G0/G1 box. By the end of the second hour of chase time, i.e., 9 h of total culture time, the nearly equal percentages of the BrdU-labeled populations (20.57% of adherent and 22.83% of control erythroblasts) had progressed through G2/M into G0/G1 phase. In the histograms at 13 h and 15 h of total culture, BrdU-labeled erythroblasts are either in G0/G1 or they have entered a second S-phase. The G0/G1 populations at 13 h and 15 h were calculated to be the total percentage of cells in the R5 box minus the percentages of BrdU-labeled erythroblasts that overlapped from S-phase. An example of this calculation for the 13 h adherent erythroblasts is: 1. At 13 h, 68.61% of total cells were BrdU-labeled and 20.47% of all erythroblasts were in the R5 box. Thus, 20.47 ÷ 68.61 = 29.83% of BrdU-labeled erythroblasts were in the R5 box and the remaining 70.17% BrdU-labeled erythroblasts were outside of the R5 box (S-phase cells). 2. Observation: Fewer cells are in S-phase at 13 h than at 7 h. Assuming that the overlap distribution of BrdU-labeled S-phase erythroblasts is similar at 13 h to that of the 7 h erythroblasts, we calculated the 13 h BrdU-labeled S-phase erythroblasts overlap into the G0/G1 box as (70.17% non-R5 S-phase at 13 h ÷ 87.14% non-R5 S-phase at 7 h) × 12.86% (overlap at 7 h) = 10.36% best estimate of overlap at 13 h. 3. If the R5 box in the 13 h histogram contained 10.36% of the BrdU-labeled adherent erythroblasts due to overlap from the S-phase erythroblasts, then the G0/G1 population in the R5 box was 29.83% - 10.36% = 19.47%. 4. Similar calculations showed that the 38.22% of BrdU-labeled control erythroblasts were in G0/G1-phase at 13 h. At 15 h, the calculations showed that 4.61% adherent BrdU-labeled erythroblasts were in G0/G1 phase, while 30.15% of control erythroblasts were in G0/G1 phase.
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