Figure 1.

Mcl-1 is required for CD4⁺ and CD8⁺, naïve and memory T cells. (a) Survival of CD4⁺ and CD8⁺ Mcl-1^f/fERCre and Mcl-1^f/f control T cells after a 3-day culture of total splenocytes in medium alone and in medium with 1 ng/ml IL-7. The percentage of live cells was assessed by PI exclusion. White bars represent cells cultured in the ethanol vehicle control (EtOH), and black bars represent cells cultured in 4-hydroxytamoxifen (4OHT). The data represent the mean survival +S.E.M. of 12–15 experiments, each performed in triplicate. Here, n.s. signifies that the P value is not significant (P≥0.05) between the samples indicated. ^**P<0.01, ^***P<0.001 versus 4OHT-treated Mcl-1^f/f control. (b) Ratio of survival in 4OHT to survival in EtOH of splenocytes cultured for 3 days in medium alone and in medium +IL-7. The ratios calculated from triplicate wells were determined for each experiment, and the bars represent the mean of the ratios + the S.E.M. of 12–15 experiments. ^***P<0.001 versus Mcl-1^f/f control. (c) The enhancement of survival by IL-7 was calculated by dividing the percent survival (as assessed by PI exclusion) in medium with IL-7 (1 ng/ml) by the survival in medium alone for each cell type in both the EtOH control and 4OHT as indicated. The bars are the mean+S.E.M. of 11–14 independent experiments. (d) Gating of CD44^hi and CD44^lo cells from total splenocyte cultures. Representative flow cytometry plots and histograms are shown. Cells were pre-gated on both live and dead cell populations by forward and side scatter. (e) Percent surviving cells by PI exclusion in total splenocyte cultures gated on CD44^hi and CD44^lo populations. Bars are the mean+S.D. of triplicate wells. (f) Mcl-1 protein expression was assessed by intracellular staining for Mcl-1 in cells cultured for 3 days with or without 4OHT. The shaded histogram indicates the isotype control. Data are representative of three experiments, each with duplicate wells