Cre-dependent excision of Mcl-1 blocks BLyS-dependent in vitro B-cell survival. (A) Excision of the Mcl-1 gene in Mcl-1f/null B cells renders the targeted B cells refractory to BLyS-mediated survival protection. Small resting B cells from donors expressing an Mx-cre transgene and heterozygous for floxed and wild-type Mcl-1 alleles (Mcl-1f/wt) or for floxed and null Mcl-1 alleles (Mcl-1f/null) were cultured for 3 days with and without 100 ng/mL rhuBLyS in the presence of 200 units of IFNα to induce Mcl-1 excision. Viability was assessed by trypan blue dye exclusion. Each point is the average of 3 determinations with statistical comparisons included: P > .05 (not significant); ***P < .001. (B) Gene dose affects the amount of Mcl-1 protein in Percoll purified resting B cells. Mcl-1 protein was determined by Western blot of serially diluted (2-fold) cell lysates prepared from B cells taken from donors heterozygous for floxed and wild-type (Mcl-1f/wt) or floxed and null (Mcl-1f/null) alleles or homozygous for floxed Mcl-1 alleles. Proteins were quantified by densitometry as described Figure 2C. Results are representative of 2 independent experiments. (C) Mcl-1 gene dose modulates the effectiveness of BLyS-dependent B-cell protection. Small resting B cells from Mcl-1f/wt, Mcl-1f/null, or Mcl-1f/f donors expressing an Mx-cre transgene were cultured for 3 days with or without 100 ng/mL rhuBLyS in the presence or absence of 200 units of IFNα. Viability was determined by trypan blue exclusion. Statistical comparisons between B cells cultured with BLyS and B cells cultured with BLyS and interferon (P > .05, not significant; **P < .01; ***P < .001). Each point is the average of 3 determinations in this representative experiment done 3 times. Nil indicates cells cultured without BLyS or IFNα.
