Fig 1: Persistence of functional respiratory CI during repression of NDUFS3(A) Immunodetection of NDUFS3 on western blots of whole-cell lysates from 143B-/- NDUFS3 cells treated with 100 ng/mL doxycycline (Dox) for 0 (untreated), 1, 2, 4, and 8 days (n = 3). NDUFS3 band intensities were quantified by densitometry and normalized to the signal of Hsp70, used as loading control. The mean values of the treated cells were referred to those of untreated (control) set to 100%. Data are means ± SD. ****p < 0.0001 treated versus untreated; one-way ANOVA with Sidak’s multiple comparisons test.(B) CI-IGA of enriched mitochondrial fractions from repressed 143B-/-NDUFS3, solubilized either with digitonin or DDM and separated by BN-PAGE. SCs include CI+III2 and respirasomes (I+III2+IV1-n).(C) Immunodetection of CI subunits on western blots of 143B-/-NDUFS3 cells, treated either with 100 ng/mL Dox for 0 (untreated), 1, 2, 4, and 8 days or simultaneously with 100 ng/mL Dox and 50 µg/mL CAP for 8 days, resolved by SDS-PAGE. ß-actin was used as loading control.(D) Immunodetection of CI subunits NDUFA12 (N-module), NDUFS2 (Q-module), NDUFS3 (Q-module), NDUFB8 (ND5-module), CIII subunit UQCRFS1, and CIV subunit MT-CO2, in western blots of mitochondrial fractions from 143B-/-NDUFS3, treated either with 100 ng/mL Dox for 0 (untreated), 1, 2, 4, and 8 days or simultaneously with 100 ng/mL Dox and 50 µg/mL CAP for 8 days, solubilized with digitonin and separated by BN-PAGE. SCs include CI+III2 and respirasomes (I+III2+IV1-n). SDHB and SDHA (CII) were used as loading controls.(E) Immunodetection of CI, CIII, and CIV subunits on western blots of mitochondrial fractions from 143B-/-NDUFS3 cells treated with 100 ng/mL Dox for 0 (untreated), 2, 4, and 8 days, solubilized with digitonin and resolved by 2D BN-PAGE. All subunits were immunodetected on the same blot. One asterisk (*) indicates a non-specific signal derived from an anti-NDUFA8 antibody with which the membrane was previously incubated. Two asterisks (**) indicate a signal derived from anti-MT-CO1.See also Figure S3.