FIGURE 5. Levels of p53 are upregulated in human B cells triggered by BCR:CD21-L + IL-4 + BAFF.

(A) Immunoblotting evidence for p53. Top: Day 3 and 5 lysates from tonsil FO cells stimulated with BCR:CD21-L ± IL-4 ± BAFF were analyzed for p53 and for β-actin loading control (following stripping and reblotting). A fresh blot and longer film exposure was used for p53 analysis T662 lysates, while a re-stripped blot with shorter film exposure was used for p53 analysis of T663 lysates. (p53 upregulation is representative of 6 experiments). (B) Comparative p53 levels within lysates of the human Ramos B cell line (expressing mutated p53) or day 3 normal human B cell cultures stimulated with BCR:CD21-L + IL-4 + BAFF. Note that the Ramos lysate was loaded at 1/20^th of the total protein as the day 3-stimulated normal B cell lysate. (C) Flow cytometric evidence for p53. Top row: histograms representing CFSE-fluorescence in cells stimulated for 4 days with BCR:CD21-L with or without IL-4 ± BAFF, prior to intracellular staining. Subsequent rows: Stimulated cells were gated on the basis of CFSE fluorescence (division status) and assessed for PE fluorescence indicative of p53 staining (line) or IgG2b control staining (tinted histogram). Shown is the ratio of mean fluorescence intensity values (RMFI) obtained when anti-p53 MFI is divided by control MFI. The proportion of viable cells following into the 0, 1, 2, and 3 division subgroups in this experiment were as follows: BCR:CD21-L only: 92, 7, 1, and 0%; BCR:CD21-L + IL-4: 72, 18, 8 and 1%; BCR:CD21-L + IL-4 + BAFF: 49, 27, 19, and 5%, respectively. (D) Pooled data from 15 experiments evaluating the relative expression of p53 within the varying division subsets. The left plot shows data expressed as RMFI; the right plot shows data expressed as % of the maximal MFI above background (Δ MFI) within each experiment. The values above the bars show the p values for level of significance between p53 expression in undivided cells and cells with the indicated division.