FIGURE 3. Replicating B cells stimulated with BCR:CD21-L + IL-4 + BAFF exhibit elevated mRNA levels of Bid and Bax, but repressed Bim mRNA.

(A) Cultures stimulated with BCR:CD21-L alone or with growth-promoting IL-4 and BAFF were harvested on day 4, mRNA isolated, and cDNA prepared with oligo(dT) primer. Quantitative PCR (q-PCR) was performed with primers specific for Bim, Bid, Bax, and β-actin. Δ Ct values for each of the above were obtained through comparison with β-actin. Values for fold difference (Δ) were obtained by comparing Δ Ct values for each pro-apoptotic mRNA in cytokine-supplemented cultures with the respective Δ Ct values in control cultures with BCR:CD21-L alone. The p values represent the significance of comparisons between Δ values in cytokine-supplemented cultures versus those in control cultures in a total of 4 replicate experiments, using two-tailed Students t test. (B) Bid mRNA levels in divided blasts are comparable to Bid mRNA in undivided blasts. For semi-quantitative RT-PCR of Bid, cells were stimulated for 5 days with BCR:CD21-L (0.01 μg/ml) + IL-4 + BAFF and sorted on the basis of CFSE fluorescence into two populations: undivided blasts and divided blasts (2–5 divisions). mRNA was isolated and cDNA prepared as above. PCR amplification was performed with Bid or β-actin-specific primers. Varying μl amounts of the amplicons were loaded onto 1.5% agarose gels and electrophoresed. PC=positive control and NC=negative control for PCR. (Similar results obtained from a 2^nd experiment evaluating levels of Bid mRNA in undivided versus divided blasts).