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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 5. Fas/FasL Signaling Is a Critical Mediator of Apoptosis Induced by Cyclin D Ablation.

Figure 5

(A) FACS analysis of Fas and FasL surface expression in MxTKO and MxCntrl bone marrow cells 2 days after ablation of D-cyclins. Note increased staining in MxTKO as compared to MxCntrl.

(B) Western blot analysis of Fas protein levels in bone marrow 2 days after ablation of D-cyclins.

(C) Upregulation of Fas and FasL mRNA levels in hematopoietic stem/progenitor cells (HSPC) 24 hours after ablation of D-cyclins (MxTKO). N=4 mice for each genotype, error bars, SD.

(D) Intracellular staining for cleaved caspase 8 in bone marrow cells 2 days after cyclin D-deletion. Note increased staining in MxTKO as compared to MxCntrl.

(E) Western blot analysis of the levels of cleaved caspase 8 in bone marrow cells of MxCntrl and MxTKO mice, three days after pI-pC injection (to delete D-cyclins in MxTKO).

(F and G) MxTKO and MxCntrl mice were injected with neutralizing anti-FasL antibody (αFasL), or with isotype control (IgG) concomitantly with administration of pI-pC (to delete D-cyclins in MxTKO mice). Two days later, hematopoietic stem/progenitor cells, HSPC (F), or hematopoietic stem cells, HSC (G) were flow sorted and stained with Annexin V and 7AAD to mark apoptotic (Annexin V+/7AAD+) cells. Note that inhibition of FasL abrogated apoptosis of HSC and HSPC triggered by ablation of D-cyclins (compare MxTKO-IgG vs. MxTKO-αFasL). (F) Shows representative staining with Annexin V 7AAD. (G) Mean percentage of Annexin V+ HSC (n=3 mice for MxCntrl and n=5 MxTKO). Error bars, SD.

See also Figure S3.