Figure 9. Bmf-deficiency delays apoptosis by cell stress associated with CAP-independent translation in primary thymocytes and SV40 MEF.

(A) SV40 MEF were deprived of serum in the absence or presence of actinomycin D or cycloheximide. Protein lysates were generated at the indicated time-points and Bmf expression was analyzed by immunoblotting. (B) Wt or bmf^−/− SV40 MEF were exposed to rapamycin or LY294002 up to 48h (upper panel). Alternatively, actinomycin D or cycloheximide was added at t=0h or after 24h of incubation with rapamycin or LY294002 (lower panel). (C) Cells lacking or expressing Bmf were treated with the indicated compounds over time. Thymocyte apoptosis was quantified by AnnexinV/PI staining (n=4/genotype). Apoptosis of MEF was assessed by sub-G1 staining and flow cytometric analysis. Bars represent means ± SE of three independent experiments. Experiments using three independent MEF clones of each genotype yielded comparable results and were therefore pooled (STS=staurosporine). Asterices * indicate statistically significant differences between wt and bmf^−/− MEF (p< 0.006 for LY294002; p< 0.001 for rapamycin). (D) Wt and Bmf-deficient mice were injected i.p. with a single dose of polyIC (100μg/mouse), or saline. After 20h, mice were sacrificed and the percentage of naïve CD8⁺CD44⁻ T cells quantified by flow cytometric analysis. Bars represent means of ± SEM of three animals per genotype and group. * indicates statistically significant differences between wt and bmf^−/− (p<0.003).