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. Author manuscript; available in PMC: 2015 Aug 11.
Published in final edited form as: Dev Cell. 2014 Jul 31;30(3):255–267. doi: 10.1016/j.devcel.2014.06.015

Figure 3. Apoptosis of Bone Marrow Cells After Ablation of D-cyclins.

Figure 3

(A and B) Mean percentage of Annexin V+ (apoptotic) bone marrow cells in MxTKO and MxCntrl mice (n=5 per genotype), at 48 and 96 hours after ablation of D-cyclins. Error bars, SD.

(B) Staining of femur sections for apoptotic cells (TUNEL). Cells were counterstained with methyl green. Note strongly increased number of apoptotic cells (TUNEL+) and paucity of viable nucleated cells (TUNEL− /methyl green+) in MxTKO bone marrow.

(C) Mean percentages of TUNEL-positive bone marrow cells analyzed by FACS 4-6 days after ablation of D-cyclins (MxTKO). Each dot corresponds to a different animal (n=7 mice per group), horizontal lines denote mean values.

(D-G) MxTKO and MxCntrl mice were injected with caspase inhibitor QVD-OPh (Cl), with vehicle only (DMSO), concomitantly with the deletion of D-cyclins, and bone marrows were collected 6 days later.

(D) Shown are representative images of hematoxylin and eosin stained sections of bone marrows. Note that ablation of D-cyclins resulted in loss of nucleated bone marrow cells (MxTKO-DMSO – upper right panel), and that treatment with caspase inhibitor partially rescued this effect (MxTKO-CI – lower right panel).

(E) TUNEL staining of femur sections to detect apoptotic cells. Cells were counterstained with methyl green. Note that treatment of animals with caspase inhibitor (CI) decreased the fraction of apoptotic cells in MxTKO mice (lower right panel) and increased the number of nucleated cells.

(F) Representative FACS profiles of bone marrow cells stained with TUNEL (to detect apoptotic cells) and with propidium iodide (PI, for cell cycle analysis) and analyzed by FACS. The percentages of apoptotic (TUNEL+) cells are marked. Note that treatment with Cl blocked apoptosis triggered by cyclin D ablation (MxTKO-DMSO vs. MxTKO-Cl). Also note that ablation of D-cyclins reduced the fraction of cells with >2N DNA content and increased the proportion of 2N cells, which is indicative of cell cycle arrest (please see also Figures S2C-S2F).

(G) Percentages of TUNEL+ cells in bone marrows of MxTKO and MxCntrl mice treated with DMSO or with caspase inhibitor QVD-OPh (Cl). TUNEL+ cells were quantified by FACS. Each dot corresponds to a different animal (n=5 for each group), horizontal lines represent mean values.

See also Figure S2.