Figure 3: Atovaquone inhibits complex III activity.

(a) The effect of atovaquone on complex I-, II-, III- or IV-dependent respiration in FaDu cells permeabilized with 0.005% digitonin. The % OCR was measured immediately after permeabilization. (b) Complex II/III activity was measured in mitochondria isolated from FaDu cells 15 min after compound addition. (c) Complex II activity was measured in mitochondria isolated from FaDu cells 15 min after compound addition. (d,e) The OCR of FaDu cells was measured for 45 min after injection of antimycin A (d) or myxothiazol (e). The % OCR post injection is shown relative to the DMSO control and normalized to the relative cell number obtained by hoechst staining at the end of the experiment. The data is representative of three independent experiments. (f,g) FaDu spheroids were treated with DMSO, antimycin A or myxothiazol for 24 h. Hypoxia was assessed by staining central spheroid sections for EF5 (green), with DAPI as a nuclear counterstain (blue). Scale bar, 600 μm. % Mean EF5 fluorescence intensity is presented relative to the DMSO controls. Hypoxia was assessed in at least five spheroids per treatment for each experiment (n=3). One-way ANOVA with Bonferroni was performed for all of the experiments (n=3, mean±s.d., ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05). ANT A, antimycin A; ATO, atovaquone; MYXO, myxothiazol.