Impaired BCR-induced survival and proliferation in Zfx-deficient B cells. (A) The expression of Zfx in wild-type FO B cells after BCR stimulation. Normalized Zfx expression levels were determined by qPCR relative to the nonstimulated sample (mean ± SD of triplicate reactions). (B) BCR-induced apoptosis of B cells from control and CD19-Cre⁺Zfx^flox/y CKO mice. Representative staining profiles of annexin V and DNA content (7-AAD) are shown for restingB cells cultured in vitro with or without anti-IgM for 24 hours. The fractions of annexin V⁺7-AAD⁻ apoptotic cells and annexin V⁺7-AAD⁺ dead cells are indicated (mean ± SD of 3 mice per group). (C) Proliferation of IgM-stimulated B cells from control and CD19-Cre⁺Zfx^flox/y CKO mice. Shown is CFSE dilution in B220⁺ B cells cultured for 3 days after anti-IgM stimulation (open histograms) or in medium alone (gray histograms). Note that the histograms are normalized by the height of the CFSE^bright quiescent cell peak, which is decreased in CKO B cells because of apoptosis; hence, the apparent increase in CKO B-cell response to anti-CD40 and LPS. Representative of 4 independent experiments. (D) Dose response of BCR-induced B-cell proliferation. B-cell proliferation was measured as above 3 or 5 days after stimulation with the indicated concentrations of anti-IgM. (E) Cell-cycle analysis of BCR-stimulated B cells from control and CD19-Cre⁺Zfx^flox/y CKO mice. Splenic B cells were cultured in vitro with anti-IgM for 48 hours and pulsed with BrdU 45 minutes before analysis. Representative staining profiles of BrdU and DNA content (7-AAD) with the percentage of cells in the indicated cell-cycle fractions are shown.