Figure 6. Restoration of B cell homeostasis in BMKO mice by genetic ablation of p53. (A) Lysates of untreated Mulefl/y (control) and Muledel/y (Mule-deficient) MEFs; control and BMKO B cells; and Mulef/y (control E14K) and Muledel/y (Mule deficient) ES cells were immunoblotted to detect total p53 protein. p53−/− MEFs, Ab specificity control. β-Tub, β-tubulin (loading control). MEF and B cell results are representative of two to three independent experiments. ES cell data are from a single experiment involving one control and three Mule-deficient clones. (B) The mRNA expression of the indicated p53 target genes was determined by quantitative real-time PCR in lysates of purified splenic B cells from mice of the indicated genotypes. The relative change in gene expression was calculated using the comparative threshold cycle method (2ΔΔCt). Results are the mean ± SD after normalization to 18S or actin mRNA and are representative of two to five independent experiments involving one to five mice per genotype. P-values (*, P < 0.05; **, P < 0.005) are from the Student’s t test of control versus BMKO or BDKO. (C, left) The development of pro–B cells (B220+IgM−cKit+; top row) and pre–B cells (B220+IgM−CD25+; middle row) in the BM of mice of the indicated genotypes was monitored by flow cytometry. The bottom row shows BrdU uptake by total splenic B cells as determined by flow cytometry. Numbers are the percentage of gated IgM− lymphocytes (top and middle), or of gated B220+ cells (bottom). (C, right) Absolute numbers of pro–B and pre–B cell cells in the BM of mice of the indicated genotypes were determined as in Fig. 2 C. Results are the mean ± SD (*, P = 0.018, Student’s t test of control versus BMKO) and are representative of two to three independent experiments involving one to two mice per genotype. (D, left) purified splenic B cells from mice of the indicated genotypes were treated with 20 µg/ml anti-IgM or 20 µg/ml anti-IgM plus 1 µg/ml anti-CD40 and [3H]-thymidine incorporation was measured at 24, 48, and 72 h after stimulation. Results are the mean± SD and are representative of two independent experiments involving one to two mice per genotype. (D, right) Splenic cellularity of mice of the indicated genotypes was determined as in Fig. 2 F. Results are the combined mean ± SD (*, P = 0.0073, Student’s t test of control versus BMKO) of four independent experiments each involving one to three mice per genotype.
Figure 6.

Restoration of B cell homeostasis in BMKO mice by genetic ablation of p53. (A) Lysates of untreated Mulefl/y (control) and Muledel/y (Mule-deficient) MEFs; control and BMKO B cells; and Mulef/y (control E14K) and Muledel/y (Mule deficient) ES cells were immunoblotted to detect total p53 protein. p53−/− MEFs, Ab specificity control. β-Tub, β-tubulin (loading control). MEF and B cell results are representative of two to three independent experiments. ES cell data are from a single experiment involving one control and three Mule-deficient clones. (B) The mRNA expression of the indicated p53 target genes was determined by quantitative real-time PCR in lysates of purified splenic B cells from mice of the indicated genotypes. The relative change in gene expression was calculated using the comparative threshold cycle method (2ΔΔCt). Results are the mean ± SD after normalization to 18S or actin mRNA and are representative of two to five independent experiments involving one to five mice per genotype. P-values (*, P < 0.05; **, P < 0.005) are from the Student’s t test of control versus BMKO or BDKO. (C, left) The development of pro–B cells (B220+IgM−cKit+; top row) and pre–B cells (B220+IgM−CD25+; middle row) in the BM of mice of the indicated genotypes was monitored by flow cytometry. The bottom row shows BrdU uptake by total splenic B cells as determined by flow cytometry. Numbers are the percentage of gated IgM− lymphocytes (top and middle), or of gated B220+ cells (bottom). (C, right) Absolute numbers of pro–B and pre–B cell cells in the BM of mice of the indicated genotypes were determined as in Fig. 2 C. Results are the mean ± SD (*, P = 0.018, Student’s t test of control versus BMKO) and are representative of two to three independent experiments involving one to two mice per genotype. (D, left) purified splenic B cells from mice of the indicated genotypes were treated with 20 µg/ml anti-IgM or 20 µg/ml anti-IgM plus 1 µg/ml anti-CD40 and [3H]-thymidine incorporation was measured at 24, 48, and 72 h after stimulation. Results are the mean± SD and are representative of two independent experiments involving one to two mice per genotype. (D, right) Splenic cellularity of mice of the indicated genotypes was determined as in Fig. 2 F. Results are the combined mean ± SD (*, P = 0.0073, Student’s t test of control versus BMKO) of four independent experiments each involving one to three mice per genotype.

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