Figure 4.

Autophagy is dispensable for B-cell proliferation and survival upon BCR stimulation. Purified splenic B cells from wild-type C57BL/6 mice (B6), Atg5^f/+ CD21cre (littermates CD21 cre) or Atg5^f/+ Mb1cre (littermates Mb1 cre), Atg5^f/− CD21 cre (CD21 cre), or Atg5^f/− Mb1 cre (Mb1 cre) were cultured without any stimulation (non-stim) or with 5 μg/ml anti-IgM in combination or not with 5 μg/ml anti-CD40 Abs or with LPS (5 μg/ml). (a) Cells from the indicated mice were stained with CFSE before culture, and proliferation was assessed by measuring the dilution of the fluorescent signal by flow cytometry after 3 days of culture. Percentages of proliferating cells are indicated in the histograms, for one representative anti-IgM/CD40 stimulation experiment, for each CD21 cre and Mb1 cre mice with their controls. (b) Mean and standard deviation (S.D.) values of the percentages of proliferating cells obtained in four independent experiments. (c) Alternatively, cells were stimulated as described and cell death was assessed by double annexin-V/7-AAD staining allowing to distinguish viable cells (annexin-V⁻7-AAD⁻) early apoptotic cells (annexin-V⁺7-AAD⁻) and late apoptotic/necrotic cells (annexin-V⁺7-AAD⁺). (d) The mean and S.D. values of viable cell proportions obtained in six independent experiments are indicated. ns, non-significant; *P<0.05, **P<0.01 (Mann–Whitney U-test)