Figure 1.

Detectable amounts of fully assembled and functional complex I are present in NDUFS3 KO cells

(A) Spectrophotometric measurements of complex I (CI) activity normalized to citrate synthase (CS). Data are means ± SD (n = 9 for 143B^−/− and n = 10 for 143B^+/+). ^∗∗∗∗p < 0.0001 according to Mann-Whitney test.

(B) CI in-gel activity (CI-IGA) of 143B^+/+, 143B^−/−, OS93 (heteroplasmic 97% mutant m.3571insC/MT-ND1), OS (homoplasmic mutant m.3571insC/MT-ND1), and Rho 0 (mtDNA depleted) samples solubilized with n-dodecyl β-D-maltoside (DDM) and separated by high-resolution clear native PAGE (hrCNE).

(C) CI-IGA of 143B^+/+ and 143B^−/− samples solubilized with digitonin and separated by BN-PAGE. SCs include CI+III₂ and respirasomes (I+III₂+IV_1−n).

(D) Immunodetection of CI, CIII, and CIV subunits on western blots of total lysates from 143B^+/+ and 143B^−/− cells lines resolved by SDS-PAGE. β-actin was used as loading control. Superfluous lanes were cropped in the UQCRC2 and relative loading control images.

(E) Immunodetection of CI, CIII, and CIV subunits on western blots of mitochondrial fractions from 143B^+/+ and 143B^−/− cell lines solubilized with digitonin and separated by 2D BN-PAGE. All subunits were immunodetected on the same blot.

(F) CI-driven ATP synthesis rates (CI-ATP) for 143B^+/+ (n = 6), 143B^−/− (n = 6), 143B^−/− treated with 50 μg/mL chloramphenicol (CAP) (n = 3), HCT116^+/+ (n = 12), HCT116^−/− (n = 8), and HCT116^−/− treated with 50 μg/mL CAP (n = 4). Rotenone-sensitive ATP synthesis rates (nmol/min × mg) were normalized to CS activity and protein content. Values are means ± SD. ^∗∗p = 0.0022, ^∗∗∗∗p < 0.0001 according to Mann-Whitney test. ND, not detected.

See also Figures S1–S3.