Respiratory chain function, measured as oxygen consumption (O₂ flux/pmol/(s x mg), mean±SEM) with high resolution respirometry on isolated liver mitochondria using SUIT protocol [19] with sequential addition of substrates, ADP, and inhibitors, showed significant changes in sick 27–29 days <i>Bcs1l^G/G</i> animals compared to control animals (<i>Bcs1l^A/A</i>).

*<p>FCCP, Carbonyl cyanide-<i>4</i>-(trifluoromethoxy)phenylhydrazone, uncoupler showing maximal capacity.</p>**<p>TMPD <i>N,N,N′,N′</i>-tetramethyl-<i>p</i>-phenylenediamine.</p

Low amount of RISP in sick <i>Bcs1l</i>^G/G mice is associated with increased amount of free pre-CIII₂ and CI.

<p>A. Representative blue native gel electrophoresis of isolated mitochondria from <i>Bcs1l</i>^G/G (G/G) and control (A/A and A/G) mice 27–28 days old. The bands of respiratory chain complexes and supercomplexes are visualized. B. In isolated liver mitochondria from <i>Bcs1l</i>^G/G, BNGE followed by Western blot shows the lack of BCS1L. Supercomplex (SC1) composition was assessed with antibodies against NDUFV1, RISP, Core1 and subunit Cox1. PDHE1α and ETFAα antibodies were used as loading controls.</p

Mitochondria from <i>Bcs1l^G/G</i> mice contain less membrane located RISP.

<p>Liver tissue from sick <i>Bcs1l</i>^G/G (G/G) and control (A/G) mice aged P26 was homogenized and subsequently separated into cytosolic, mitochondrial and mitochondrial membrane fractions. These fractions were analyzed by SDS PAGE and Western blot detecting CIII subunits Core1 and RISP, CI subunit NDUFA9, and CIV subunit Va. Porin and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as markers for mitochondrial membrane and cytosol, respectively, and as loading controls.</p

Increased expression of CI and CII subunits in liver tissue of sick <i>Bcs1l</i>^G/G animals.

<p>The mRNA expression levels of <i>Bcs1l</i> and subunits of respiratory chain complexes were analyzed with quantitative PCR. In homozygotes, the subunits of CI and that of CII were significantly up-regulated in comparison with control animals.</p

The main supercomplex contains CI, pre-CIII and fully assembled CIII₂.

<p>Isolated liver mitochondria from P14, P20 and P29 <i>Bcs1l</i>^G/G (G/G) and control (A/A and A/G) mice were analyzed with two-dimensional run with BNGE/SDS PAGE and Western blot. Antibodies for complexes are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086767#pone-0086767-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086767#pone-0086767-g003" target="_blank">3</a>. The ratio of RISP/Core1 was clearly smaller in P29 homozygotes than in control animals.</p

Age dependent decrease of RISP in young <i>Bcs1l</i>^G/G mice.

<p>A. Subunits of all complexes were assessed after SDS PAGE of isolated mitochondria from <i>Bcs1l</i>^G/G (G/G) and wild type (A/A) young mice using the following antibodies; SDHA (CII), CVa (CV), Core1 (CIII), NDUFA9 (CI), RISP (CIII), and Subunit Va (CIV). Pyruvate dehydrogenase (PDHE1α) and electron-transfer-flavoprotein, alpha polypeptide (ETFAα) were used as loading controls. B. Supercomplexes in homozygous and wild type young mice were investigated with BNGE and Western blot using antibodies detecting subunits NDUFV1, RISP, Core1 and subunit Cox1 (CIV).</p

2D-BNGE with Western blot detection showing supercomplex composition in mitochondria of the three mouse strains studied.

<p>Antibodies against CIII (RISP) and CIV (COX1) subunits were initially used and after stripping the antibody against Core1. After a second stripping the antibody against CI (NDUFA9) was used. Supercomplexes are indicated by vertical boxes. (A) In the 129/Sv wild-type mouse, CIV was present in two supercomplexes. (B) Both CIV containing supercomplexes were present in wild-type mice (<i>Bcs1l</i>^<i>A/A</i>) of 129/Sv:C57BL/6 mixed background strain (MB with <i>SCAFI</i>^{<i>long/short</i>}). In mutant (<i>Bcs1l</i>^<i>G/G</i>) mice, CIV was decreased in the respirasome CI/CIII₂/CIV and more abundant in the formation of pre-CIII₂/CIV. (C) In backcrossed (BC) C57BL/6 congenic strain (with <i>SCAFI</i>^{<i>short/short</i>}), CIV was very faintly present in supercomplexes even when exposure time was increased compared to MB. In homozygotes (G/G) of both backgrounds, a CI₂/CIII₂ supercomplex was found and free CI was more abundant than in wild-type (assessed with densitometry: 26% in 129/Sv, 27% vs. 49% in MB and 48% vs. 56% in BC wild-type and homozygotes, respectively), as also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168774#pone.0168774.g002" target="_blank">Fig 2</a>.</p

<i>SCAFI</i> genotype and age at end-stage disease (age at sacrificing) of homozygous (<i>Bcs1l</i>^<i>G/G</i>) mice in different genetic backgrounds.

<p><i>SCAFI</i> genotype and age at end-stage disease (age at sacrificing) of homozygous (<i>Bcs1l</i>^<i>G/G</i>) mice in different genetic backgrounds.</p

BNGE analysis.

<p>Comparison of isolated liver mitochondria from <i>Bcs1l</i> mutant homozygotes (G/G) and wild-types (A/A) of 129/Sv:C57BL/6 mixed background (MB), C57BL/6 backcrossed (BC) strain and the 129/Sv strain. Four supercomplex bands (SC) are visible in wild-type animals in MB and 129/Sv strains, one of the bands not found in C57BL/6 animals. In homozygotes (G/G) in both MB and BC strains, free complex I (CI) is more abundant than in wild-type animals (A/A), and free complex III (CIII) is only present as a pre-complex (PreCIII₂).</p

<i>SCAFI</i> allele status and its effect on supercomplex assembly in <i>Bcs1l</i> mutant mice (G/G) of different genetic backgrounds.

<p>Wild-types (A/A) and <i>Bcs1l</i> mutant mice (G/G) of 129/Sv:C57BL/6 mixed background (MB) and C57BL/6 background (BC) were analyzed and a wild-type of the 129/Sv strain (129/Sv) was used as a control. (A) Agarose gel electrophoresis of SCAFI genotyping PCR products showing the long (<i>SCAFI</i> long) and short (<i>SCAFI</i> short) alleles in mice of different genetic backgrounds. (B) Western blot analysis of SCAFI protein and ETFAα (electron transfer flavoprotein alpha) as loading control in liver tissue lysate showing reduced level of SCAFI protein in mixed background (MB) mice and no detectable protein in C57BL/6 mice (BC). (C) Respiratory chain complexes and supercomplexes were separated on BNGE and detected by Western blot using the antibodies indicated. Molecular weights are indicated on the right. Note that the COX1 antibody required an extended exposure time for detection in supercomplexes. ETFAα was used as a loading control. NDUFV1 antibody shows in mutant (G/G) mice abundant free CI, in all mice CI/CIII_2, and in wild-type MB and 129/Sv that this is overlapping with the band for CI/CIII₂/CIV. Core1 shows pre-CIII₂ and CIII₂, and RISP fully assembled CIII₂ indicating presence of CIII in all supercomplexes of 129/Sv and wild-type MB animals, but mainly in supercomplex CI/CIII₂ in mutant (G/G) mice of both backgrounds. CIV antibody shows a clear band in respirasome in 129/Sv mouse, but of varying intensity in MB wild-type mice in relation to the loading control (ETFAα).</p