FIGURE 7:

Overexpression of ANTs in ant2^TALEN cells rescues ADP/ATP exchange and improves bioenergetic efficiency. (A) Relative mRNA level of ANTs in ANT overexpressors and the relevant wt and ant2^TALEN vector controls. Data were analyzed by the ΔΔC_T method, represented as mean fold change (2^-ΔΔCT) ± SEM (n ≥ 3) relative to ANT expression in wt transfected with empty vector, which was set to 1. (B) Whole-cell extracts (50 μg) from wt, ant2^TALEN, and ant2^TALEN clones overexpressing various ANT isoforms immunoblotted with ANT1- and ANT2/ANT3-specific antibody. GRP75 and β-actin served as loading controls. (C) Densitometric analyses of ANT2/ANT3 (left) or ANT1 (right) expression in cell extracts from B. Expression relative to wt ± SEM (n ≥ 7). (D) Mitochondria (100 μg) from wt, ant2^TALEN, and ant2^TALEN clones overexpressing various ANT isoforms were fused with liposomes internally preloaded with 5 mM unlabeled ADP. Uptake of [¹⁴C]ADP (nM/mg protein/min ± SEM; n = 6) in the presence and absence of 60 μM CATR. (E) ANT-specific activity was calculated from D by subtracting CATR-sensitive exchange from ADP/ATP transport in the absence of inhibitor and expressed as a mean percentage of wt (100%) ± SEM (n ≥ 3). (F) OCR and (G) calculated basal, maximal, proton-leak OCR (pmol O₂/min ± SEM; n ≥ 21), degree of coupling, spare respiratory capacity (as percentage of wt + v), and ATP production of ant2^TALEN cells overexpressing ANT1, ANT2, or ANT3 (± SEM; n ≥ 21). Note that the values obtained from vector-transfected wt and ant2^TALEN cells are the same as under 0 μM BKA treatment in Figure 6C. (H) Basal, maximal, and proton-leak OCR, as well as ATP production, degree of coupling, and spare respiratory capacity, obtained after overnight incubation with the indicated concentrations of BKA (μM).