Figure 6.
Lentivirus-mediated expression of human BCL-2 can rescue loss of MCL-1 in early erythropoiesis. TER119-depleted fetal liver cells from E12.5 Mcl1F/F Rosa-ERCreT2+ or Mcl1F/wt Rosa-ERCreT2+ littermate controls were infected with zsGreen-tagged lentivirus encoding human cDNAs (BCL2, MCL1) or EGFP for 16 hours. Cultures were pulsed with 4-OHT for the next 16 hours and then media were changed to EPO-containing media to support erythroid differentiation. (A-B) Flow cytometric analysis of viral vector expression in fetal liver cells; UTC, untransduced control cells. (A) Percentage of zsGreen+ cells in the singlet gate. (B) Mean fluorescence intensity (MFI) of zsGreen signal (n ≥ 3 biological replicates with 3 embryos each). Data are mean ± standard error of the mean (SEM). *P < .5, **P < .01, ***P < .01, ****P < .001, 1-way analysis of variance (ANOVA) with α = 0.05. (C) Fetal liver cells were infected with the indicated virus, pulsed with 4-OHT as indicated, harvested, and analyzed by PCR. Top row is floxed Mcl1. Second row is deleted Mcl1, where the presence of band indicates deletion. Third row is the human lentivirus insert encoding MCL1. Fourth row is the human lentivirus insert encoding BCL2. (D-E) Counts of lentivirus-infected cells following erythroid-differentiation protocol. Cells were left untreated (D) or pulsed with 4-OHT (E), harvested, and counted. Counts are represented as fold change relative to empty vector (n ≥ 3 biological replicates with 3 embryos each). Data are mean ± SEM. ****P < .001, 1-way ANOVA with α = 0.05. (F-H) Cells infected with the indicated viruses were differentiated, harvested, and analyzed by flow cytometry. CD45− cells falling into the live gate were analyzed by expression of CD71 and TER119. (F) Representative plots of empty vector–expressing cells left untreated (upper right panel) or pulsed with 4-OHT (lower right panel); graph shows cumulative differentiation stage data. MCL1-expressing (G) and BCL2-expressing (H) cells (n ≥ 3 biological replicates with 3 embryos each), Data are mean ± SEM. F/wt, Mcl1F/wt; F/F, Mcl1F/F; LV, lentivirus; Tam, tamoxifen.

Lentivirus-mediated expression of human BCL-2 can rescue loss of MCL-1 in early erythropoiesis. TER119-depleted fetal liver cells from E12.5 Mcl1F/F Rosa-ERCreT2+ or Mcl1F/wt Rosa-ERCreT2+ littermate controls were infected with zsGreen-tagged lentivirus encoding human cDNAs (BCL2, MCL1) or EGFP for 16 hours. Cultures were pulsed with 4-OHT for the next 16 hours and then media were changed to EPO-containing media to support erythroid differentiation. (A-B) Flow cytometric analysis of viral vector expression in fetal liver cells; UTC, untransduced control cells. (A) Percentage of zsGreen+ cells in the singlet gate. (B) Mean fluorescence intensity (MFI) of zsGreen signal (n ≥ 3 biological replicates with 3 embryos each). Data are mean ± standard error of the mean (SEM). *P < .5, **P < .01, ***P < .01, ****P < .001, 1-way analysis of variance (ANOVA) with α = 0.05. (C) Fetal liver cells were infected with the indicated virus, pulsed with 4-OHT as indicated, harvested, and analyzed by PCR. Top row is floxed Mcl1. Second row is deleted Mcl1, where the presence of band indicates deletion. Third row is the human lentivirus insert encoding MCL1. Fourth row is the human lentivirus insert encoding BCL2. (D-E) Counts of lentivirus-infected cells following erythroid-differentiation protocol. Cells were left untreated (D) or pulsed with 4-OHT (E), harvested, and counted. Counts are represented as fold change relative to empty vector (n ≥ 3 biological replicates with 3 embryos each). Data are mean ± SEM. ****P < .001, 1-way ANOVA with α = 0.05. (F-H) Cells infected with the indicated viruses were differentiated, harvested, and analyzed by flow cytometry. CD45− cells falling into the live gate were analyzed by expression of CD71 and TER119. (F) Representative plots of empty vector–expressing cells left untreated (upper right panel) or pulsed with 4-OHT (lower right panel); graph shows cumulative differentiation stage data. MCL1-expressing (G) and BCL2-expressing (H) cells (n ≥ 3 biological replicates with 3 embryos each), Data are mean ± SEM. F/wt, Mcl1F/wt; F/F, Mcl1F/F; LV, lentivirus; Tam, tamoxifen.

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