Figure 4. Combined Loss of Crebbp and Ep300 Blocks Cell Proliferation.

(A) WB analysis of Crebbp and Ep300 expression in Ficoll-separated splenic B cells of the indicated genotypes, cultured ex vivo in the presence of αCD40 and IL-4 for 4 days. Analysis of H3K27Ac and H3K18Ac monitors for the functional effects of Crebbp and/or Ep300 loss and is quantified on the bottom. Tubulin and total H3 serve as loading control for whole-cell and chromatin extracts, respectively.

(B) Representative histogram plots showing the number of cell divisions in cultured B cells from the indicated genotypes, measured on day 4 after labeling with the CellTraceViolet reagent (live cells gate).

(C) Quantification of the data shown in (B) (mean ± SD; n = 3 mice per genotype).

(D) Cell growth in the same cells, measured by enzymatic activity and expressed as fold changes relative to day 0 (mean ± SD; n = 3 mice per genotype);

(E and F) Analysis of cell viability, assessed on the basis of the percentage of dead cells in the forward scatter versus side scatter (FSC/SSC) (E) and the percentage of AnnexinV⁺ cells (F). Data are from one experiment where all genotypes were simultaneously analyzed and are representative of at least two independent experiments performed with subsets of genotypes (n = 3 each) that gave analogous results and were combined for statistical analyses. Note that the ex vivo assay is associated with an intrinsic elevated cell death. Bars represent mean ± SD.

Only statistically significant p values are indicated. *p < 0.05, **p < 0.01; Student’s t test.