Figure 1

Mcl-1 transgene expression in lpr mice. (a) Lymphocytes from Mcl-1tg/lpr mice are resistant to apoptosis triggered through either the intrinsic or extrinsic pathways. Sorted CD4⁺CD8⁺ DP thymocytes from WT, Mcl-1tg, lpr and Mcl-1tg/lpr mice were cultured in vitro at 50 × 10³ cells in 100 μl for 48 h in medium alone (left panel), medium containing 32 ng/ml Fc-FasL (centre panel) or 1 μg/ml etoposide (right panel). Viability was determined as the proportion of cells negative for PI uptake and annexin V surface staining, normalized to viability at 0 h. Viability following treatment with Fc-FasL and etoposide are given relative to untreated cells to show stimulus-specific apoptosis. Data represent average response of three independent mice per genotype±S.E.M. (b-h) Expression of endogenous and tg Mcl-1 protein in diverse haemopoietic cell types. Western blot analysis of lysates of (b) CD4⁺ T cells (CD4⁺CD8^–), (c) CD8⁺ T cells (CD4^–CD8⁺) and (d) unusual T cells (TCRβ⁺CD4^–CD8^–B220⁺) sorted from spleen; (e) Pre B cells (CD19⁺IgM^–IgD^–) sorted from bone marrow; (f) B cells (CD19⁺) sorted from spleen; (g) granulocytes (Mac1⁺Gr1⁺) and (h) macrophages (Mac1⁺Gr1^–) sorted from bone marrow. Genotypes and mouse identification numbers are indicated. Lysates prepared from whole spleen of lpr mouse #641 and Mcl-1tg/lpr mouse #637 were run on each gel to facilitate cross-comparison between different cell types. The solid line indicates electronic removal of two lanes. Each gel was probed with anti-Mcl-1 and anti-FLAG antibodies; because of the presence of the FLAG epitope tag, tg Mcl-1 protein is larger than endogenous Mcl-1. Representative blots of n=1–4 experimental replicates. Refer to Supplementary Figure S1 for quantification of blots; sorted myeloid cells and spleen cells appear to contain degraded Mcl-1 making interpretation of relative levels of endogenous and tg Mcl-1 ambiguous