Fig. 2: ETFDH participates in the Q cycle.
a, CIII enzymatic activity in isolated mitochondria from CRL and ETFDH-ko myocytes. The left panel shows absorbance (A) changes over time. In the right panel, CIII activity is presented as mU per mg of mitochondrial protein. n = 3. Ant A was used as a CIII inhibitor. b, ΔΨm in CRL and ETFDH-ko myocytes in the presence or absence of 1 µM Ant A. n = 3. c–e, Mitochondrial (c, e) and cytosolic (d) ROS in CRL, ETFDH-ko and cDNA-rescued ETFDH-ko (+ETFDH) myoblasts in the presence or absence of Rot, Mal and Ant A. The upper scheme in e illustrates the site of action of each inhibitor. n = 3. mitoROS, mitochondrial ROS. f, Absorbance of Cyt b (λ = 565 ± 5 nm) before and after DTT administration in isolated mitochondria of CRL (black trace) and ETFDH-ko (red trace) myocytes. n = 3. g, Relation between Q10H2/Q10 ratio, Q9H2/Q9 ratio and mitochondrial ROS in CRL and ETFDH-ko myoblasts. n = 3. h, ETFDH structure highlighting two highly conserved amino acids in the Q-binding domain: Y271 and G273. i,j, Representative western blot analysis of ETFDH protein levels (i) and mitochondrial ROS (j) in CRL and ETFDH-ko myoblasts expressing native (ETFDH-wt) or mutated (ETFDH-Y271A,G273E) ETFDH protein. n = 3. k, Schematic of AOX functioning (top) and representative western blot of ETFDH expression (bottom) in CRL and ETFDH-ko cells expressing or not expressing AOX. l–o, Mitochondrial ROS (l), ΔΨm (m), respiratory profile (n) and CIII activity (o) in CRL and ETFDH-ko cells expressing or not expressing AOX. n = 3. Results are shown as the mean ± s.e.m. of the indicated n. NS, not significant. *, **, ***, ****P < 0.05, 0.01, 0.001 and 0.0001 when compared with CRL by two-tailed Student’s t test (a), one-way ANOVA with Tukey’s test (c, d, j, l–o), and two-way ANOVA with Šidák’s test (b) or Tukey’s test (e).
