PRELI localizes in mitochondria, alters the Δψ_m, and induces mitochondrial ROS production. (A) PRELI localizes in mitochondria and reduces Δψ_m. HeLa cells were stained for pFlag-PRELI (transfected cell indicated by white arrow). Mitochondria were visualized with MitotrackerRed CMXRos. Intensity profiles (middle panel) visualize FITC (PRELI overexpression) and Mitotracker fluorescence intensities in these cells. CD4⁺ cells (right panel) were nucleofected with pIRES-H2K^k (control) or PRELI overexpression vector (black bars) and subsequently sorted. Cells were also nucleofected with scramble or PRELI-siRNAs (). Subsequently, the cells were activated (with α-CD3 0.1 or 0.5 μg/well and α-CD28) for 24 hours, stained with Mitoprobe DilC₁⁵  and analyzed by flow cytometry. Two separate cell populations with high and low Mitoprobe DilC₁⁵  intensities seen in the flow cytometric analysis depict cells with active and reduced membrane potentials, respectively (histograms not shown). Thus, the percentage of cells with reduced membrane potential stands for the proportion of cells with low Mitoprobe DilC₁⁵  intensity in a sample. Bars represent the mean values plus or minus SEM of 7 independent experiments. (B) PRELI induces mitochondrial ROS production. Overexpressed pFlag-PRELI (Alexa 568, red) and mitochondrial ROS production stained with DHR123 (green). CD4⁺ cells (right panel), nucleofected and activated as in panel A, were stained with DHR123 and analyzed by flow cytometry. Bars represent the average fold changes plus or minus SEM of ROS intensity in PRELI overexpression or PRELI-siRNA samples compared with the corresponding control sample (pIRES-H2K^k or scramble). The values of the control samples were set as 1. Overexpression and siRNA data are obtained from 7 and 4 independent experiments, respectively. (A,B) Δψ_m and ROS were measured from living cell populations according to forward scatter/side scatter (FSC/SSC) plots (overexpression experiments) or total cell population (siRNA experiments). Statistical significances were calculated using the 2-tailed paired t test. (C) Representative levels of PRELI protein in overexpression and knockdown experiments. In overexpression experiments the peripheral blood CD4⁺ cells, transfected with pIRES-H2K^k-PRELI or pIRES-H2K^k control vector, were enriched 16 hours after the nucleofection and subsequently activated for 24 hours. In PRELI knockdown experiments, the cells nucleofected with scrambled- or PRELI-siRNA oligos were incubated for approximately 24 hours and subsequently activated for 24 hours. Samples were harvested for Western blotting before (in overexpression experiments after the enrichment) and after the activation. Vertical lines have been inserted to indicate a repositioned gel lane. OE indicates overexpression.