Figure 3
Figure 3. c-Myb is required for lymphoid progenitors to respond to IL-7 and TSLP. (A) Limiting dilution analysis of LSK cells isolated from the bone marrow of 6- to 8-week-old c-Myb+/+ and c-MybPlt4/Plt4 mice. Cultures were performed on OP9 or OP9-DL1 stromal cells in the presence of IL-7 and Flt3L. *P < .025. Data represent the mean ± SEM of 5 samples per genotype. (B) Limiting dilution analysis of CLPs isolated from the bone marrow of 6- to 9-week-old c-Myb+/+ and c-MybΔmb1/Δmb1 mice. Cultures were performed on OP9 or OP9-DL1 stromal cells in the presence of IL-7 and Flt3L. The difference in B-cell precursor frequency was not significant (P > .025), probably because of incomplete deletion of the floxed allele; see panel C. Data represent the mean ± SEM of 2 to 3 samples per genotype. (C) PCR genotyping of B-cell colonies generated from c-MybΔmb1/Δmb1 CLPs. CLPs were isolated from c-Mybfl/fl or c-MybΔ/fl mice carrying the mb1Cre allele. After 8 days of culture on OP9 stromal cells, individual B-cell colonies were harvested, and DNA was extracted for genotyping at the c-Myb locus. The deleted (Δ), floxed (fl), and wild type (+, derived from the OP9 cells) alleles are indicated. Data are representative of 26 colonies analyzed. Sizes in base pairs are indicated on the right. (D) Limiting dilution analysis of pro-B cells (B220+CD19+c-kit+) isolated from the fetal liver of c-Myb+/+, c-MybPlt4/+, and c-MybPlt4/Plt4 e15.5 to e16.5 embryos. Cultures were performed on OP9 stromal cells in the presence of either IL-7 or TSLP. *P < .025; **P < .005; ***P < .001. Data represent the mean ± SEM of 2 to 8 embryos per genotype. Only 2 small colonies were observed from c-MybPlt4/Plt4 pro-B cells stimulated with IL-7; hence, the number in the graph represents an overestimate of precursor frequency. The precursor frequency in panels A, B and D was defined as the inverse of the number of cells per well at which more than 37% of the wells lacked a colony. (E) Intracellular flow cytometry for phosphorylated STAT5 (pSTAT5) after IL-7 stimulation of c-Myb+/+ and c-MybPlt4/Plt4 bone marrow. Plots are gated on B220+CD43+ pro-B cells and are representative of 4 mice per genotype. The red line indicates cells without IL-7; the blue line indicates cells stimulated with IL-7.

c-Myb is required for lymphoid progenitors to respond to IL-7 and TSLP. (A) Limiting dilution analysis of LSK cells isolated from the bone marrow of 6- to 8-week-old c-Myb+/+ and c-MybPlt4/Plt4 mice. Cultures were performed on OP9 or OP9-DL1 stromal cells in the presence of IL-7 and Flt3L. *P < .025. Data represent the mean ± SEM of 5 samples per genotype. (B) Limiting dilution analysis of CLPs isolated from the bone marrow of 6- to 9-week-old c-Myb+/+ and c-MybΔmb1/Δmb1 mice. Cultures were performed on OP9 or OP9-DL1 stromal cells in the presence of IL-7 and Flt3L. The difference in B-cell precursor frequency was not significant (P > .025), probably because of incomplete deletion of the floxed allele; see panel C. Data represent the mean ± SEM of 2 to 3 samples per genotype. (C) PCR genotyping of B-cell colonies generated from c-MybΔmb1/Δmb1 CLPs. CLPs were isolated from c-Mybfl/fl or c-MybΔ/fl mice carrying the mb1Cre allele. After 8 days of culture on OP9 stromal cells, individual B-cell colonies were harvested, and DNA was extracted for genotyping at the c-Myb locus. The deleted (Δ), floxed (fl), and wild type (+, derived from the OP9 cells) alleles are indicated. Data are representative of 26 colonies analyzed. Sizes in base pairs are indicated on the right. (D) Limiting dilution analysis of pro-B cells (B220+CD19+c-kit+) isolated from the fetal liver of c-Myb+/+, c-MybPlt4/+, and c-MybPlt4/Plt4 e15.5 to e16.5 embryos. Cultures were performed on OP9 stromal cells in the presence of either IL-7 or TSLP. *P < .025; **P < .005; ***P < .001. Data represent the mean ± SEM of 2 to 8 embryos per genotype. Only 2 small colonies were observed from c-MybPlt4/Plt4 pro-B cells stimulated with IL-7; hence, the number in the graph represents an overestimate of precursor frequency. The precursor frequency in panels A, B and D was defined as the inverse of the number of cells per well at which more than 37% of the wells lacked a colony. (E) Intracellular flow cytometry for phosphorylated STAT5 (pSTAT5) after IL-7 stimulation of c-Myb+/+ and c-MybPlt4/Plt4 bone marrow. Plots are gated on B220+CD43+ pro-B cells and are representative of 4 mice per genotype. The red line indicates cells without IL-7; the blue line indicates cells stimulated with IL-7.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal