Functional cloning of Bcl10 as a factor preventing BCR-induced apoptosis. (A) Scheme for selection of cDNAs that protect WEHI-231 lymphoma B cells against anti-IgM-induced growth arrest and apoptotic death (also see “Functional cloning of Bcl10 as an antiapoptotic factor”). (B) A Bcl10 cDNA recovered from the screen was inserted into a puromycin-resistant retrovirus. Infected WEHI-231 cultures were selected for puromycin-resistance for 5 days and then treated with anti-IgM (45 hours) to test the prosurvival activity of the cDNA. The number of viable (PI-excluding) cells was determined by flow cytometry and compared with that of the unstimulated control culture as a percentage. Squares indicate Bcl10-expressing retrovirus; diamonds, vector only. Similar results were obtained with a second Bcl10 cDNA recovered from the screen (not shown). (C) A short-term survival assay was performed on WEHI-231 cells infected with a retrovirus encoding a bicistronic message of Bcl10 open reading frame (ORF; without untranslated regions)-IRES-GFP. Infected cells were incubated for 1 day to allow Bcl10 expression and then stimulated with varying doses of anti-IgM for 45 hours. The number of viable cells was determined as in panel B. ▪ indicates GFP⁺ cells; ✕ , GFP^- cells. The ability of the retroviral construct to express Bcl10 (HA-tagged) was verified by immunoblotting of transfected Bosc23-cell lysates (not shown). The noninfectants were GFP^- cells in the infected cultures. Infection with a vector-derived virus had no effect on BCR-induced apoptosis (data not shown). (D) Bcl10 also conferred survival advantage to CH31 cells. Cells were infected by Bcl10-IRES-GFP retrovirus and examined similarly to panel C, but with or without a saturating concentration of anti-IgM (10 μg/mL) (vector control, IRES-GFP). Values shown are averages of triplicate determinations ± SD (P < .005 for the comparison of anti-IgM-treated cells with Bcl10 versus anti-IgM-treated cells without Bcl10).