Fig 1: CI assembly in WT and ?4-CYB cells solubilized with DDM and in modulated NDUFAF2 levels (related to Fig 5) Complexome profiles of the cI structural modules found in both cell lines in the two reciprocal labeling experiments. The graphs plot the average of relative peptide peak intensities along the lane corresponding to the subunits of each module, setting the maximum to 1 versus the molecular mass, calculated using the individual complexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and ?4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cI modules. The x-axis values were the slice number (1-64), and the y-axis values were the relative peptide intensity. The plotted values are mean ± SD (n = 2). Two-way ANOVA with Sidak's multiple comparisons test **P = 0.0072 (Q-module); **P = 0.0029 (N-module); *P = 0.0102.A Myc-DDK (FLAG) tagged version of NDUFAF2 (AF2Myc-DDK) was stably expressed in both WT and ?4-CYB cybrids by transfection of the cloned cDNA in the pCMV6-Entry mammalian expression vector (Origene Cat#: RC207387). As negative controls, the two cell lines were also transfected with the empty pCMV6-Entry vector (EV), providing the resistance to neomycin but no expression of other relevant proteins.Complex I-IGA assay performed in the NDUAF2-overexpressing (AF2Myc-DDK) and negative control (EV) cell lines after solubilizing the samples with digitonin and separating them on BNGE.Complex I enzymatic activity assay performed in the NDUAF2-overexpressing (AF2Myc-DDK) and negative control (EV) cell lines. Results are expressed as mean ± SD (n = 3 biological replicates). No significant differences (ns) in cI activity were found between the NDUFAF2-overexpressing WT or ?4-CYB cells and their corresponding negative control (one-way ANOVA with Tukey's multiple comparisons test).NDUFAF2 expression was knocked down both WT and ?4-CYB cybrids by transfection of two different siRNAs targeting the NDUFAF2 transcript (AF2 siRNA1 and AF2 siRNA2, Sigma-Aldrich). Sigma's siRNA Universal Negative Control #1 was used in the experiments as well.Complex I-IGA assay performed in the cells transfected with both siRNAs and the negative control (CT(-) siRNA), after solubilizing the samples with digitonin and separating them on BNGE.Complex I enzymatic activity assay performed in the cells transfected with both siRNAs and the negative control cell lines. Results are expressed as mean ± SD (n = 3 biological replicates). No significant differences (ns) in cI activity were found between the silenced WT or ?4-CYB cells and their corresponding negative control (one-way ANOVA with Tukey's multiple comparisons test). Source data are available online for this figure.