Figure 5.
Removal of apoptosis rescues erythropoiesis defect caused by deletion of Mcl1. (A) Schematic diagram of transplant studies indicating lethally irradiated CD45.1+ recipients and 4 groups of CD45.2+ donors: C57BL/6 wild-type, Mcl1F/F Rosa-ERCreT2, BaxF/FBak−/−Rosa-ERCreT2, and Mcl1F/FBaxF/FBak−/− Rosa-ERCreT2 (n = 5-11 recipients per donor group; see "Materials and methods"). These 4 groups were further subdivided into untreated or tamoxifen treated, for a total of 8 treatment groups. (B) Kaplan-Meyer survival plot of recipient animals following tamoxifen treatment. (C) Immunoblot of recipient spleens (2 lanes each) following BM transplant and tamoxifen-mediated deletion of floxed alleles. (D) BM from vehicle or tamoxifen-treated recipients was differentiated into erythroblasts on fibronectin-coated plates in EPO-containing media. Cells were labeled with CD45, CD71, and TER119 and then analyzed by flow cytometry; CD45− fraction is shown divided into developmental stages S0 through S5. Bars represent mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with α = 0.05 was used to compare each group vs wild-type (WT). (E) Methylcellulose colony-forming assays were performed using recipient total BM; BFU-Es were grown on MethoCult GF M3434 (contains EPO), whereas CFU-Ms and CFU-GMs were grown on MethoCult GF M3534 (without EPO). (F-G) Recipient BM was harvested and differentiated into erythroblasts on fibronectin-coated plates as in Figure 4. (F) BM from mice that did not receive tamoxifen during the transplant study was differentiated with or without 4-OHT, as indicated. Cells were labeled with CD45, CD71, and TER119 and then analyzed by flow cytometry; representative plots of the CD45− fraction are shown. (G) Ex vivo–differentiated BM was labeled with CD45, CD71, TER119, and Annexin V antibodies and analyzed by developmental stage (n = 5-11 recipient animals per donor group). Data are mean ± SEM. *P < .05, **P < .1, ***P < .01, ****P < .001, 2-way ANOVA with α = 0.05. DKO, double knockout; F/F, Mcl1F/F; Tam, tamoxifen; TKO, triple knockout; WT, wild-type.

Removal of apoptosis rescues erythropoiesis defect caused by deletion of Mcl1. (A) Schematic diagram of transplant studies indicating lethally irradiated CD45.1+ recipients and 4 groups of CD45.2+ donors: C57BL/6 wild-type, Mcl1F/F Rosa-ERCreT2, BaxF/FBak−/−Rosa-ERCreT2, and Mcl1F/FBaxF/FBak−/− Rosa-ERCreT2 (n = 5-11 recipients per donor group; see "Materials and methods"). These 4 groups were further subdivided into untreated or tamoxifen treated, for a total of 8 treatment groups. (B) Kaplan-Meyer survival plot of recipient animals following tamoxifen treatment. (C) Immunoblot of recipient spleens (2 lanes each) following BM transplant and tamoxifen-mediated deletion of floxed alleles. (D) BM from vehicle or tamoxifen-treated recipients was differentiated into erythroblasts on fibronectin-coated plates in EPO-containing media. Cells were labeled with CD45, CD71, and TER119 and then analyzed by flow cytometry; CD45− fraction is shown divided into developmental stages S0 through S5. Bars represent mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with α = 0.05 was used to compare each group vs wild-type (WT). (E) Methylcellulose colony-forming assays were performed using recipient total BM; BFU-Es were grown on MethoCult GF M3434 (contains EPO), whereas CFU-Ms and CFU-GMs were grown on MethoCult GF M3534 (without EPO). (F-G) Recipient BM was harvested and differentiated into erythroblasts on fibronectin-coated plates as in Figure 4. (F) BM from mice that did not receive tamoxifen during the transplant study was differentiated with or without 4-OHT, as indicated. Cells were labeled with CD45, CD71, and TER119 and then analyzed by flow cytometry; representative plots of the CD45− fraction are shown. (G) Ex vivo–differentiated BM was labeled with CD45, CD71, TER119, and Annexin V antibodies and analyzed by developmental stage (n = 5-11 recipient animals per donor group). Data are mean ± SEM. *P < .05, **P < .1, ***P < .01, ****P < .001, 2-way ANOVA with α = 0.05. DKO, double knockout; F/F, Mcl1F/F; Tam, tamoxifen; TKO, triple knockout; WT, wild-type.

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