A20-deficiency amplifies B-cell responses. (A) Expression levels of the respective B-cell activation marker after overnight stimulation with LPS or αCD40. The dot-plots are representative of 3 to 6 independent experiments. (B) [³H]Thymidine incorporation during a 10-hour pulse 48 hours after stimulation of B-cell cultures with αIgM, αCD40, LPS, or CpG. Data are mean ± SD of triplicate measurements. The experiment was repeated with similar results. (C) Cell cycle profile analysis by propidium iodide staining of B cells 2 days after stimulation with the indicated mitogens. Percentages of dead (sub G₀) and live cells are indicated at the top of each histogram. The distribution within the cell cycle was calculated with the FlowJo software Version 8.7.3 using the Watson model (values do not add up to 100%). Data are means of 2 independent experiments. (D) Assessment of proliferation by the carboxyfluorescein succinimidyl ester dilution assay: histograms represent carboxyfluorescein succinimidyl ester intensities 3 days after stimulation. The tables under each histogram show the proliferation index (Prol. Index: average number of divisions of the proliferating cells), the percentage of dividing cells (% Divided: the proportion of cells that initially started to divide), and the division index (Div. Index: average number of divisions of all cells); values were calculated with the FlowJo software. Data represent the means of 5 independent experiments, and bold values are significantly different (P < .05) from wild-type according to 1-way analysis of variance analysis. (E) Evaluation of IL-6 production in overnight activated B cells by ELISA (top panels), intracellular FACS (middle panels), and real-time PCR (bottom panels). For intracellular FACS, B cells were stimulated for 3 days. cDNA was quantified relative to porphobilinogen deaminase. Data are mean ± SD of 3 independent experiments. (F) Quantification of TNF production by overnight-stimulated B cells via ELISA. Data are mean ± SD of 3 independent experiments.