Figure 5

Prominent apoptosis, increased turnover, and decreased expression of Bcl-2, A1, and Bcl-X _L in AUF1-deficient FO B cells. (a-b) Prominent apoptosis (arrows) seen in splenic B cell follicles from (a) AUF1^-/- and (b) irradiated RAG1^-/- mice reconstituted with AUF1^-/- bone marrow. Apoptosis was confirmed by cleaved caspase-3 staining. Scale bar = 0.1 mm. (c) Analysis of in vivo B cell turnover. Mice were fed BrdU for 7d, and incorporated BrdU was determined by flow cytometric analysis. The percentage of BrdU-positive FO (CD21^intCD24^lo) or MZ (CD21^hiCD23^lo) B cells from the spleens of wild-type (white) and AUF1^-/- (black) mice was plotted; n = 3 per time point. (d) Relative Bcl-2, A1, Bcl-X_L, BIM mRNA levels in sorted FO B cells from AUF1^-/- and wild-type mice were determined by qRT-PCR. Values were normalized to cyclophilin levels and depicted as mean ± SD, n = 4. (e) Four-color flow cytometric analysis was performed to identify intracellular Bcl-2 protein levels in FO and MZ B cells. Histograms depict the intensity of wild-type (bold) and AUF1^-/- (shaded) cells stained with Bcl-2 antibody or wild-type stained with isotype-matched control antibody (thin line). Quantification of Bcl-2 expression. The normalized values (MFI for Bcl-2 - MFI of isotype-matched control) for AUF1^-/- cells (FOB 11.9, MZB 9.6) were plotted as a percentage of wild-type values (FOB 16.9, MZB 9.0). Graph represents mean ± SD, n = 4. * p < 0.05. (f-g) Sorted FO B cells were cultured in media alone or with anti-CD40 (10 μg/ml) for times indicated and (f) the percent of live cells was determined by 7-AAD exclusion or (g) Bcl-2 and A1 mRNA levels were determined 24 hrs post culture. Values represent means ± SD for three to four mice in each group.