Obesity-related hormone outcomes at baseline and week-24 comparing participants in the exercise group (n = 15) and control group (n = 15).

<p>HMW, high molecular weight; IL-6, interleukin-6; TNF-α, tumour necrosis factor-alpha; CRP, C-reactive protein; HOMA, homeostasis model assessment.</p><p>^a Change in exercise group versus change in control group, adjusted for baseline value (ANCOVA).</p><p>^b Backtransformed from natural log; expressed as relative ratio.</p><p>Obesity-related hormone outcomes at baseline and week-24 comparing participants in the exercise group (n = 15) and control group (n = 15).</p

Body composition, fitness, strength and gastro-oesophageal reflux outcomes at baseline and week-24 comparing participants in the exercise group and control group.

<p>^a Change in exercise group versus change in control group, adjusted for baseline value (ANCOVA).</p><p>^b Measured using Gastro-oesophageal reflux disease impact scale.</p><p>Body composition, fitness, strength and gastro-oesophageal reflux outcomes at baseline and week-24 comparing participants in the exercise group and control group.</p

Participant flow diagram.

<p>Participant flow diagram.</p

Baseline characteristics of participants allocated to the exercise and control groups.

<p>Data are mean ± SD, median (25^th, 75^th percentile) or n (%).</p><p>^a measured using International Physical Activity Questionnaire (recreational physical activity).</p><p>^b measured using Gastro-oesophageal reflux disease impact scale.</p><p>Baseline characteristics of participants allocated to the exercise and control groups.</p

Investigating a role for the Bateman domain in IMPDH clustering.

<p>(A) Superimposed structure of IMPDH2 (1B3O; yellow) with IMPDH1 (1JCN; red). Ligands have been removed for clarity. N labels the N-terminus. (B) Micrographs of CHO cells transiently expressing HA-IMPDH proteins (as labelled), treated with vehicle (control) or 1 µM MPA for 4 h. Cells were fixed and labelled for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Representative of at least four independent experiments. (C) Representative micrographs of CHO cells transiently expressing IMPDH1/IMPDH2 chimera (as labelled). Cells were fixed and stained for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Scale bars = 10 µm. (D) Table shows qualitative scoring of chimera subcellular distribution pattern according to the similarity to IMPDH1 (macrostructure formation) or IMPDH2 (diffuse) distribution. Assignments were based on three independent experiments. Shown by the schematics are the IMPDH1 (red) and IMPDH2 (yellow) regions of chimera constructs, with internal numbering referring to IMPDH1 sequence boundary and the striped boxes indicating the CBS dimer.</p

IMPDH activity in the presence of nucleotides.

<p>Activity of purified His-IMPDH proteins following 20 min pre-incubation with 1 mM or 5 mM nucleotides (ATP, AMP, XMP) and normalised to control, no addition, activity. Shown is the mean ± SEM (n = 3, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> to the control activity).</p

Immuno-EM of IMPDH localisation.

<p>Representative electron micrographs of CHO cells treated with (A) vehicle or (B and C) 2 µM MPA for 4 h. Cells were fixed, processed and labelled with anti-panIMPDH antibody and gold-labelled anti-mouse secondary antibody, as outlined in methods. Plasma membrane (PM), Nucleus (N) and mitochondria (M) are indicated. Scale bars = 0.5 µm.</p

R224P mutation affects ATP mediated protease protection and affects spontaneous clustering.

<p>(A) Representative Coomassie stained gel of a protease protection assay with His-IMPDH1 proteins. INM stands for IMP, NAD and MPA. (B) Quantitation of remaining full-length protein from protease protection assay presented as percent protection. Shown is the mean ± SEM and the number of experiments (n) is indicated below the graph. (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> the control (digested), # <i>p</i><0.01 <i>cf.</i> IMPDH1. (C) Representative micrographs of CHO cells transiently expressing HA-IMPDH1, HA-IMPDH1 R224P or HA-IMPDH1 D226N. Cells were fixed and stained for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Representative of at least four independent experiments. Scale bar = 10 µm. (D) CHO cells transiently expressing HA-IMPDH proteins, as indicated, were treated with either vehicle, 2 µM MPA for 4 h or 2 µM MPA for 4 h and supplemented with 100 µM guanosine for the final 2 h. Cells were fixed and stained with anti-HA antibody. From random fields, 50–80 labelled healthy cells were counted, in a blinded manner, and the classification of the subcellular distribution of the protein classified as diffuse (blue), in spicules (red) or macrostructures (green). Representative of two independent experiments.</p

Nucleotides protect IMPDH in an isoform-specific manner via the Bateman domain.

<p>(A) Representative Coomassie stained SDS-PAGE gel of a protease protection assay experiment, performed as outline in methods, with His-IMPDH2. INM stands for IMP, NAD and MPA. Molecular weight marker sizes are indicated. (B) Quantitation of remaining full-length protein from protease protection assay with His-IMPDH2 and His-IMPDH1 presented as percent protection. Shown is the mean ± SEM and the number of experiments (n) is indicated below the graph. (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> the control (digested), # <i>p</i><0.01 IMPDH1 <i>cf.</i> IMPDH2). (C) Representative gel of a protease protection assay experiment with His-Core2 protein. (D) Quantitated results as per B. Shown is the mean ± SEM from five independent experiments. (***<i>p</i><0.001 <i>cf.</i> the control (digested), ## <i>p</i><0.001 IMP, NAD and MPA <i>cf.</i> adenosine nucleotides).</p

MPA induced clustering of HA-IMPDH2-GFP in live cells.

<p>Images are maximum intensity projections from confocal z-series of live HeLa HA-IMPDH2-GFP cells treated with 2 µM MPA. Selected frames, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051096#pone.0051096.s010" target="_blank">Video S1</a>, are presented with the time captured relative to the addition of MPA recorded at the top right (h∶mm∶ss). Data is representative of three independent experiments. (A) Field of cells with low HA-IMPDH2-GFP expression, which is initially diffuse throughout the cytosol and with time HA-IMPDH2-GFP clusters into spicules and macrostructures. Scale bar = 20 µm. (B) Selected frames within subset region shown in A, corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051096#pone.0051096.s011" target="_blank">Video S2</a>, which highlight emergence of macrostructures, movement while undergoing a 180° clockwise rotation of in the X-Y plane (indicated by yellow arrow) and end-to-end fusion of spicules (indicated by yellow asterisk). Scale bar = 10 µm.</p