p63 regulates B-cell survival. (A-C) DNA screening of the wild-type (+/+), heterozygotic (+/−), and p63^−/− (−/−) fetal liver cells (as previously described¹² ) that were injected into irradiated mice (A). Splenocytes derived from the chimeric mice were stimulated with or without anti-CD74, and TAp63 or Bcl-2 mRNA levels were determined by RT-PCR (B). Annexin staining was performed on freshly isolated splenocytes (time 0) and on cells cultured for 12 or 24 hours. Histograms show the annexin-positive population of B220⁺ cells. The results presented are representative of 4 different experiments (C). (D) Primary CD74^−/− B cells were transfected with empty plasmid, CD74-ICD, TAp63α, TAp63γ, or ΔNp63 constructs as described in “Cell transfection.” After 48 hours, the IgD⁺ population was analyzed by FACS. The results presented are the mean of 3 independent experiments. Error bars represent SD. The percentage increase of IgD⁺ cells was calculated by subtracting staining of empty expression plasmid from the percentage of IgD⁺ cells in each treatment, and dividing it into the same increased value, multiplied by 100%. The results presented are representative of 5 different experiments. The intensity of the p63 band was calculated as described in Figure 2.