Functional characterisation of <i>EF-G2mt</i> equivalent SNPs in the yeast <i>MEF2</i> gene.

<p>(A) Growth of wild-type, <i>mef2</i> deletion strain and <i>mef2</i> mutants in yeast medium containing either glucose as the carbon source or the non-fermentable carbon source glycerol. Cells from exponentially growing cultures were serially diluted and 5 µl of each dilution spotted onto each plate. Growth was assessed after 72 hours incubation at 30°C. (B) Percentage cell viability of yeast <i>mef2</i> mutants in 110 µM atorvastatin relative to viability of the wild-type strain following exposure to atorvastatin for 5 days. Data represent mean ± SEM (n = 3). A one-way ANOVA followed by a Dunnett's multiple comparison test was used to compare the mean percentage viability of the <i>mef2</i> variants to that of the wild-type. ***<i>P</i><0.001.</p

Growth of siRNA transfected RD cells in glucose and galactose medium.

<p>At 24 hours post-transfection, cells were seeded into wells of a 96-well plate in DMEM medium containing 10% fetal bovine serum and either 4.5 g/L glucose (left panel) or 4.5 g/L galactose (right panel). Cell proliferation was determined daily using a luminescent cell viability assay. An untransfected cell line and the rho0 cell line were included as controls. Cell proliferation is shown as a percentage of the maximum cell growth (100%) of the untransfected control. Error bars represent the mean ± SEM for three independent measurements.</p

Visualisation of mitochondrial membrane potential.

<p>Cells were stained with MitoTracker Red CMXRos and observed using a laser scanning confocal microscope. To better visualise mitochondrial structure within the <i>mef2</i> deletant, cells were stained with a 10× concentration of MitoTracker Red. Scale bar represents 5 µm.</p

<i>In silico</i> model of the human EF-G2mt protein.

<p>(A) Model depicting four of the five amino acid variants that were functionally characterised in this study. (B) Five newly discovered variants that have yet to be functionally characterised. Helices are shown as ribbons, beta-sheets are depicted as flat broad arrows and loops and coils appear as thin tubes. The five protein domains of the EF-G2mt protein are distinguished by different colours. Domain I, the GTP binding domain is shown in blue, domain II is purple, domain III is orange, domain IV is cyan and domain V is green. Model was visualised using the Visual Molecular Dynamics program (VMD), version 1.8.7 (University of Illinois).</p

Oxygen consumption rates of <i>mef2</i> mutant cultures.

<p>Figure depicts the rate of oxygen consumption per minute per 30 mL of yeast culture at an OD₆₀₀ of 0.2. Data represent mean ± SEM (n = 3). A one-way ANOVA followed by a Dunnett's multiple comparison test was used to compare the mean oxygen consumption rate of <i>mef2</i> variants to that of the wild-type. ***<i>P</i><0.001.</p

EF-G2mt protein variants.

<p>Alignment of the protein amino acid sequence of the human EF-G2mt protein with the yeast Mef2 protein. Dark shaded areas represent conserved amino acid residues and grey shaded areas represent semi-conserved residues. <i>EF-G2mt</i> SNPs that are semiconserved in yeast <i>MEF2</i> are shown in italics and fully conserved SNPs are depicted in bold. The five alleles selected for functional characterisation are outlined. The five EF-G2mt protein domains are represented below the alignment. Global alignment of protein sequences was performed using Lalign and the BioEdit sequence alignment editor was used to generate the graphical representation.</p

IC₅₀ values for <i>EF-G2mt</i> silenced cells exposed to atorvastatin.

<p>IC50 values of <i>EF-G2mt</i> silenced cells were compared to their respective non-targeting siRNA transfected cell lines using the extra sum-of-squares F-test.</p>*<p><i>P</i><0.05.</p