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1 Supplemental material THE JOURNAL OF CELL BIOLOGY Mourier et al., Figure S1. Size and mitochondrial content in Mfn1 and Mfn2 knockout hearts. (A) Body weight of male and female control (white bars), Mfn1 (gray bars), and Mfn2 (black bars) conditional knockout mice at 20 wk of age (n = 8 of each genotype). Error bars indicate ±SEM. (B) Heart weight/body weight ratio of male and female control (white bars), Mfn1 (gray bars), and Mfn2 conditional knockout (black bars) mice at 20 wk of age (n = 8 of each genotype). Error bars indicate ±SEM. (C) Quantitative PCR determination of mtdna (mtdna: 16S, ATP6) to nuclear DNA (ndna: 18S, bact) ratios in heart from control (white bars), Mfn1 (gray bars), and Mfn2 conditional knockout (black bars) mice at 20 wk of age (n = 4 of each genotype). Error bars indicate ±SEM. (D) Electron microscopy analysis of fixed heart tissue samples from control (white bars), Mfn1 (gray bars), and Mfn2 conditional knockout (black bars) mice at 20 wk of age (n = 4 of each genotype). Representative images are shown on the left and quantification of mitochondrial mass is shown on the right. (E) Representative picture of a single adult cardiomyocyte expressing mito-yfp in combination with TMRM staining. The TMRM channel is also provided in MFN2 maintains coenzyme Q levels Mourier et al. lookup table (LUT) scale to highlight the different intensities in mitochondrial potential within themitochondrial same cell. Bar, 20 µm. The right panels represent line-scan analyses of TMRM intensities from regions of interest (ROI1 and ROI2). (F) Brightfield (BF) images of the cells shown in Fig. 1 A. Bar, 20 µm. S1

2 Figure S2. Respiratory chain activity and phospholipid composition of mitofusin knockout heart mitochondria. (A) Size distribution of mitochondria isolated from control (white bars, n = 75 mitochondria analyzed), Mfn1 (gray bars, n = 104 mitochondria analyzed), and Mfn2 knockout (black bars, n = 147 mitochondria analyzed) hearts, analyzed by electron cryo-tomography. (B) Oxygen consumption of heart mitochondria from control (white bars, n = 14 independent experiments), Mfn1 (gray bars, n = 13 independent experiments), and Mfn2 conditional knockout (black bars, n = 16 independent experiments) animals at age 60 wk. ***, P < (C) Quantification of levels of mitochondrial cytochromes performed by redox absorbance spectra of mitochondrial extracts from control (white bars), Mfn1 (gray bars), and Mfn2 conditional knockout (black bars) animals (n = 5 independent experiments for each genotype). (D) Quantification of the phospholipid composition of isolated mitochondria from control (white bars), Mfn1 (gray bars), and Mfn2 knockout (black bars) hearts, analyzed by TLC experiments as presented in Fig. 4 A (n = 4 animals per genotype). (E) Peroxidic yield, i.e., the ratio between the hydrogen peroxide production rate and the oxygen consumption flux, assessed in heart mitochondria from control (white bars), Mfn1 (gray bars), and Mfn2 conditional knockouts (black bars) in the presence of succinate and rotenone in phosphorylating (phospho) and nonphosphorylating (non phospho) conditions (n = 5 independent experiments per genotype). Error bars indicate ±SEM. S2

3 Figure S3. The coenzyme Q deficiency is associated with a loss of enzymes and metabolites of the terpenoid synthesis pathway. (A) Cholesterol quantification performed on all heart tissue extracts or mitochondria isolated from control (n = 5 independent experiments), Mfn1 (n = 5 independent experiments), and Mfn2 heart knockout (n = 5 independent experiments). Error bars indicate ±SEM. (B and C) Functional annotation enrichment analysis of proteins down-regulated in Mfn2 knockout MEFs (B) and analysis of proteins down-regulated in Mfn1 knockout MEFs (C). The association of a protein with the respective enriched category is indicated by the blue coloring; the absence of association is shown with white. Only functional categories which pass the 0.05 FDR adjusted ( Benjamini ; green bars) and nonadjusted p-values (red bars) are presented. The broken line shows the 0.05 threshold p-value. (D) Graph presenting the comparison of ratios of protein abundance in Mfn1 knockout and control MEFs. Data presented in B, C, and D were determined using SILAC labeling in two independent batches (biological replicates). MFN2 maintains mitochondrial coenzyme Q levels Mourier et al. S3

4 Figure S4. Analyses of MEFs grown on glucose or galactose media. (A) Western blot analyses of steady-state levels of mitofusin proteins in control, Mfn1 knockout, and Mfn2 knockout MEFs. (B) Quantitative RT-PCR analysis of MFN1 and -2 transcript levels in control, Mfn1 knockout, and Mfn2 knockout MEFs. (C) Growth curves of control (white circles), Mfn1 knockout (gray circles), and Mfn2 knockout (black circles) MEFs grown on DMEM glucose medium. (D) Growth curves of control (white circles), Mfn1 knockout (gray circles), and Mfn2 knockout (black circles) MEFs grown on DMEM galactose medium. Error bars indicate ±SEM. ***, P < S4

5 Figure S5. Coenzyme Q deficiency in the absence of Mfn2 is independent from mitochondrial network morphology. (A and C) Representative confocal pictures of control (A) and Mfn2 knockout MEF (C) cells transfected with a construct encoding GFP alone or GFP and a microrna for knockdown of Drp1. Mitochondrial morphology was analyzed by staining for the mitochondrial marker Tom20. Insets with lower magnification of the same area show the presence of the GFP reporter. Bars, 10 mm. (B and D) Coenzyme Q9 and Q10 quantification in control (B) and Mfn2 knockout (D) MEFs. The bars show both absolute values (raw) as wells as the values obtained after normalization over the transfection efficiency (normalized). Error bars indicate ±SEM. (E) Representative pictures showing merge (MtGreen TMRM) and single channel (TMRM) of a Mfn2 knockout MEF. Bar, 10 µm. (F) Representative example of the difference between masks obtained for the cell in E after analysis with the Image Calculator plug-in from ImageJ, as described in the Materials and methods. The black pixels highlight the areas presenting a significant reduction of TMRM signal. (G) Table summarizing the link between mitochondrial activity, bioenergetics properties, and mitochondrial network morphology in different MFN2 MEF genetic maintains knockouts mitochondrial and knockdown. coenzyme (H) Table Q summarizing levels Mourier the parameters al. used to analyze the different metabolites by mass spectrometry. MRM transitions used for quantification are shown in red. S5

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