Figure 3: Neither overexpression nor knockdown of Alex3 impairs the bioenergetic mitochondrial parameters or Ca²⁺ handling.

HEK293T cells (a,b) were transfected with Alex3 or pcDNA3–control, and the oxygen consumption (a) and mitochondrial DNA copy number (b) were measured. There were no differences between control (pcDNA3 or non-transfected (NT) cells) and Alex3-overexpressing cells (a,b). When the uncoupling protein CCCP was added to the medium, oxygen consumption increased but again the differences were not statistically significant. (c,e) Hippocampal neurons were infected with pWPI (control; black bars) and pWPI–Alex3 (purple bars) in overexpression experiments, and shRNAi–control (black bars) or shRNAi–Alex3 (purple bars) in knockdown experiments. After 7 days, we measured mitochondrial DNA copy number (c), COX and citrate synthase activity (d) and mitochondrial membrane potential (mit. memb. pot.) (e) (JC1 assay, ratio between red (Fl2) and green (FL1) fluorescence). The values were normalized against values from non-infected neurons (NI). No significant differences were found in any of the above parameters. The values show mean±s.e.m. from three independent experiments. (f,g) Mitochondrial Ca²⁺ uptake in HEK293T cells transfected with mitochondria-targeted aequorin (see Methods) together with Alex3 (or pcDNA3 in the control). (f) Intact cells were stimulated by ATP+CCh (100 μM each) to trigger maximum IP3-induced Ca²⁺ release from the ER. Because of extremely close coupling between ER and mitochondria, much of this released Ca²⁺ is taken up by the mitochondrial Ca²⁺ uniporter. There were no significant differences between control (pcDNA3) and Alex3 conditions. (g) The mitochondrial uptake from intracellular-like medium was directly measured in cells permeabilized with 60 μM digitonin for 1 min in Ca²⁺-free solution (see Methods). Then perfusion was switched first to 0.1 μM Ca²⁺, which is insufficient to activate the mitochondrial uniporter, and then to 1 μM Ca²⁺, which promotes some mitochondrial Ca²⁺ uptake. Uptake was similar in control and Alex3-transfected cells in all experimental conditions. Data represent mean±s.e.m. from 4–5 independent experiments.