Figure 5.
Figure 5. Inducible inactivation of c-myb reveals its requirement for maintenance of c-Kit expression in erythroid progenitors. (A) Schematic representation of c-myb alleles: wild type (wt), targeted floxed (F), and recombined by Cre-recombinase (Δ). Exons are shown as black boxes. Arrowheads represent loxP sites. The probe (P) used in hybridization to Southern blots is indicated by the gray box. H indicates HindIII. (B) Detection of Cre-mediated recombination of the c-mybF allele in cultured erythroid progenitors. Fetal liver cells from E13 c-myb–/F/Cre embryos were cultured as described in Figure 3 for 8 days to enrich for erythroid progenitors. IFN–α-A (2000 U/mL) was then added to the cultures and washed off at 24 hours. Genomic DNA was harvested at the indicated time points and digested with HindIII. Southern blot analysis was performed with probe P, and signals were quantified by phosphorimaging. The deletion rate of the c-mybF allele is represented as the ratio of intensities of the c-mybΔ signal to the constant c-myb– signal (right panel). (C) Flow cytometric analysis of c-Kit surface expression on cultured c-myb–/F/Cre and c-myb–/F cells in response to IFN treatment. Day 8 fetal liver cultures were treated with IFN–α-A (2000 U/mL) for 24 hours (open histogram) or were left untreated (filled histogram). At 24 or 48 hours, cells were stained with α-c-Kit-PE and analyzed by flow cytometry. (D) Real-time RT-PCR analysis of c-Kit mRNA expression in response to c-myb inactivation. Cells were treated with IFN–α-A, as described, and RNA was harvested at the indicated time points and reverse transcribed. Real-time PCR was performed as described in “Materials and methods.” (Left) Ratio of normalized relative expression levels from samples with IFN to samples without IFN was calculated for c-myb–/F/Cre and c-myb–/F separately. The average of at least 6 replicates is shown. ANOVA (2 × 2) was performed, and P values are given for the interaction term (cell type × treatment). (Right) Absolute expression (normalized to 8-hour control IFN) of c-Kit RNA determined by real time RT-PCR is shown for the c-myb–/F and c-myb–/F/Cre samples with or without IFN at 8 and 24 hours. Data for the 24-hour time point from a second independent experiment are illustrated on the right. (E) Real-time RT-PCR analysis of c-Kit mRNA expression in c-myb+/+ and c-mybKD/KD CFU-E stage cells sorted from E14 fetal liver. Normalized relative expression levels are shown. Error bars represent SEM. (F) c-myb inactivation does not lead to a general induction of differentiation. Enriched erythroid progenitors from c-myb–/F/Cre and c-myb–/F fetal livers were treated with IFN–α-A (2000 U/mL) for 24 hours (open histogram) or were left untreated (filled histogram) and subsequently were cultured for another 24 hours. Cells were stained with α-TER119 (PE) and analyzed by flow cytometry. (G) Cells were treated with IFN–α-A, as described in panel F, and were collected on cytospins at the indicated time points. After o-dianisidine/hematoxylin staining, 4 fields were counted for the proportion of hemoglobin-positive cells. The ratio of the frequencies of Hb+ cells in samples with IFN to samples without IFN is represented. Error bars represent the SEM.

Inducible inactivation of c-myb reveals its requirement for maintenance of c-Kit expression in erythroid progenitors. (A) Schematic representation of c-myb alleles: wild type (wt), targeted floxed (F), and recombined by Cre-recombinase (Δ). Exons are shown as black boxes. Arrowheads represent loxP sites. The probe (P) used in hybridization to Southern blots is indicated by the gray box. H indicates HindIII. (B) Detection of Cre-mediated recombination of the c-mybF allele in cultured erythroid progenitors. Fetal liver cells from E13 c-myb–/F/Cre embryos were cultured as described in Figure 3 for 8 days to enrich for erythroid progenitors. IFN–α-A (2000 U/mL) was then added to the cultures and washed off at 24 hours. Genomic DNA was harvested at the indicated time points and digested with HindIII. Southern blot analysis was performed with probe P, and signals were quantified by phosphorimaging. The deletion rate of the c-mybF allele is represented as the ratio of intensities of the c-mybΔ signal to the constant c-myb– signal (right panel). (C) Flow cytometric analysis of c-Kit surface expression on cultured c-myb–/F/Cre and c-myb–/F cells in response to IFN treatment. Day 8 fetal liver cultures were treated with IFN–α-A (2000 U/mL) for 24 hours (open histogram) or were left untreated (filled histogram). At 24 or 48 hours, cells were stained with α-c-Kit-PE and analyzed by flow cytometry. (D) Real-time RT-PCR analysis of c-Kit mRNA expression in response to c-myb inactivation. Cells were treated with IFN–α-A, as described, and RNA was harvested at the indicated time points and reverse transcribed. Real-time PCR was performed as described in “Materials and methods.” (Left) Ratio of normalized relative expression levels from samples with IFN to samples without IFN was calculated for c-myb–/F/Cre and c-myb–/F separately. The average of at least 6 replicates is shown. ANOVA (2 × 2) was performed, and P values are given for the interaction term (cell type × treatment). (Right) Absolute expression (normalized to 8-hour control IFN) of c-Kit RNA determined by real time RT-PCR is shown for the c-myb–/F and c-myb–/F/Cre samples with or without IFN at 8 and 24 hours. Data for the 24-hour time point from a second independent experiment are illustrated on the right. (E) Real-time RT-PCR analysis of c-Kit mRNA expression in c-myb+/+ and c-mybKD/KD CFU-E stage cells sorted from E14 fetal liver. Normalized relative expression levels are shown. Error bars represent SEM. (F) c-myb inactivation does not lead to a general induction of differentiation. Enriched erythroid progenitors from c-myb–/F/Cre and c-myb–/F fetal livers were treated with IFN–α-A (2000 U/mL) for 24 hours (open histogram) or were left untreated (filled histogram) and subsequently were cultured for another 24 hours. Cells were stained with α-TER119 (PE) and analyzed by flow cytometry. (G) Cells were treated with IFN–α-A, as described in panel F, and were collected on cytospins at the indicated time points. After o-dianisidine/hematoxylin staining, 4 fields were counted for the proportion of hemoglobin-positive cells. The ratio of the frequencies of Hb+ cells in samples with IFN to samples without IFN is represented. Error bars represent the SEM.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal