PRELI induces the mitochondrial apoptosis pathway in Th cells and is partly inhibited by IL-4. (A) Overexpression of PRELI induces mitochondria-mediated apoptosis and this is partly counteracted by IL-4. CD4⁺ cells nucleofected with pIRES-H2K^k or PRELI overexpression vector were sorted and activated as in Figure 2A, cultured with or without IL-4 for 24 hours, and stained and analyzed by flow cytometry. (B) Knockdown of PRELI and IL-4 treatment has additive effects on the mitochondrial apoptosis pathway. CD4⁺ cells nucleofected with scramble or PRELI-siRNAs were cultured, stained, and analyzed as in panel A. (A,B) Cell viability = PI-negative cells. Bars represent the mean values plus or minus SEM of 4 to 5 independent experiments, except that caspase-3 was measured in 2 independent overexpression experiments that included IL-4 treatment. In addition, caspase-3 was measured in 5 independent PRELI overexpression experiments without the IL-4 treatment (data not shown). In these experiments PRELI significantly increased the proportion of cells with active caspase-3 (*P < .05). In graphs of annexin V, bars represent the average fold changes plus or minus SEM of annexin V intensity between the untreated control sample and the other samples. The values of the untreated control samples were set as 1. Δψ_m was measured from living cell populations according to FSC/SSC plots (overexpression experiments) or total cell population (siRNA experiments). The percentage of cells with reduced Δψ_m was determined as described in Figure 2A. Statistical significances were calculated using the 2-tailed paired t test. OE indicates overexpression.