Figure 2.

Mitochondrial membrane potential is affected by queuine deficiency. HeLa cells were cultured for 48 h in the absence or presence of 1 µM queuine, as indicated. (A–B) Total DNA was extracted and examined by quantitative PCR (upper panel) for the mitochondrial encoded cox1 gene (black trace) and the nuclear encoded ndufv1 gene (red trace) producing amplicons of 189 bp and 176 bp respectively (inset figure; M, mitochondrial; N, nuclear) and the ratio of mitochondrial number to nuclear DNA was determined. (C–E) Protein extracts were examined for citrate synthase and cytochrome c oxidase activity, and the ratio of both values calculated to provide an index of ETC activity to mitochondrial volume. (F) Following fixation, dehydration, and embedding, HeLa cells were sectioned and counterstained with uranyl acetate and lead citrate for analysis by TEM (JOEL 1210 microscope) at 15,000× magnification (top row) and 25,000× magnification (bottom row). (G) Mitochondrial membrane potential was examined by the uptake of the lipophilic cation TPMP⁺. Energy dependent TPMP⁺ uptake, in the absence (left) or the presence (right) of the F₁F_O ATP synthase (F_O-subunit) inhibitor oligomycin, was expressed as the accumulation ratio per mg of protein relative to TPMP⁺ concentration in the medium according to the equation (TPMP.mg^-1 protein/TPMP.µL^-1 supernatant). Mean (± s.d.) for triplicate samples. n ≥ 2. *p < 0.05, **p < 0.01, t-test.