PR3 Regulates the Apoptosis of Hematopoietic Stem and Progenitor Cells
(A) Gating strategy to identify macrophage frequency in BM after treatment with PBS or clodronate liposomes. Numbers denote the frequency of specific cell subsets among live singlets.
(B) Quantification of BM macrophage frequency in WT and Prtn3−/− mice after liposome injection (n = 13–18 per group).
(C) Frequency of viable (Annexin V− 7-AAD−), early apoptotic (Annexin V+ 7-AAD−), and late apoptotic (Annexin V+ 7-AAD+) LSK cells in WT and Prtn3−/− mice after BM macrophage depletion. Numbers denote the frequency of cells among LSK cells (left panel). Quantification is shown in the right panel (n = 12–13 per group).
(D) Caspase-3 activation in LSK cells using a cell-permeable, fluorescent caspase-3 substrate. Numbers denote the frequency of LSK cells with active caspase-3 (left panel). Quantitative analysis is shown in the right panel (n = 5 per group).
(E) Apoptosis in sorted LSK cell cultures at 36 hr. Rate of apoptosis is calculated relative to the initial number of cells. %Apoptosis = (initial number of cells – total number of Annexin V− 7-AAD− cellular events detected)/initial number of cells (n = 8 per group).
(F) Analysis of caspase-3 activity in sorted LSK cells using a luminescence-based assay at 12 or 36 hr post culture. Data were normalized to the average luminescence value of WT cells for each independent experiment (n = 7–11 per group).
See also Figures S5 and S6.