Quantitative analysis of mIFNα2 and mIFNα2-dAb biodistribution.

<p>Quantitative analyses of ¹¹¹In labelled mIFNα2 and fusion protein levels were carried out 3 hours after intravenous administration in BALB/c mice via tail vein injection of approximately 0.5 MBq radiolabeled compound. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057263#s3" target="_blank">Results</a> show accumulation of radiolabelled mIFNα2-dAb fusions in mouse liver is considerably higher than that observed with mIFNα2. Data also shows increased hepatic accumulation of mIFNα2-DOM26h-196-61 compared to mIFNα2-DOM26h-V_HD2 isotype control. Error bars shown represent standard deviation of the mean, n = 4 (n = 3 in the case of mIFNα2).</p

Surface plasmon resonance analysis of ASGPR specific dAbs and mIFNα2-dAb fusion proteins.

<p>Murine ASGPR H1 antigen immobilised on CM5 chip surface was used to analyse binding kinetics of DOM26h-196-61 and mIFNα2-DOM26h-196-61 injected over the chip surface at a constant flow rate of 50 µl.min⁻¹. mIFNα2-V_HD2 isotype control was also injected over the chip surface as a negative control for antigen binding.</p

<i>In vitro</i> activity of mIFNα2 formatted as dAb fusions.

<p>Activity of the mIFNα2-dAb fusion proteins was tested in the B16-Blue™ assay and compared to unfused mIFNα2 standard. Error bars are not visible as they are smaller than the data points, but represent standard error of the mean of 3 independent experiments. mIFNα2-DOM26h-196-61 (dashed line, closed circles) and mIFNα2-V_HD2 isotype control (dotted line, closed diamonds) showed comparable activity to the H₆-mIFNα2 standard (solid line, closed squares), with only minor increases in the EC₅₀.</p

Genotype-specific senescence responses in HCT116 isogenic cell lines.

<p>Genotype-specific senescence responses in HCT116 isogenic cell lines.</p

Network analysis suggests a more robust senescence phenotype is induced in the presence of activating <i>KRAS</i>^<i>G13D</i> mutation.

<p>Signalling network built out from ECT2 as a seed object using the Shortest Paths algorithm in GeneGo from Metacore. Gene expression data from HCT116 parental and HCT116 KRAS^+/- cells was overlaid on this network. Green lines represent HCT116 parental-specific paths, dark blue represent HCT116 KRAS^+/- specific paths, light blue represent common paths. Red circles next to icons reflect up-regulation, while blue circles reflect down-regulation in expression profiles. Shading intensity indicates fold change (minimum 2-fold). Icons indicate protein functional classes.</p

HCT116 isogenic cell panel.

<p>HCT116 isogenic cell panel.</p

Senescence signalling and proliferation effects induced by siRNAs in the presence of common cancer-associated gene mutations.

<p>(a) Heat-map drawn in Tableau desktop representing hit (green) and miss (red) for mean nuclear area and SAβGal for the top 16 siRNA hits in HCT116 parental and isogenic derivatives. Hits were classed as ≥ 2/3 wells in ≥ 2/3 independent experiments passing the cut-off of mean scrambled control + 3 standard deviations. Values in heat-map boxes represent the number of repeat experiments in which at least 2/3 siRNA gave results greater than scrambled mean + 3SD. Mean nuclear area per well (μm²) in (b) HCT116 parental, (c) HCT116 p53 null, (d) HCT116 p21 null, (e) HCT116 KRAS^+/- and (f) HCT116 PIK3CA^+/- ranked in descending order for each cell line. Mean nuclear area per well (μm²) for triplicate wells in 3 independent transfections are represented as box whisker plots generated in Tableau desktop. Boxes represent the 25^th– 75^th percentile of the data. Median level is shown as a colour change within the box. Positive (CDK1) and negative (Scrambled) siRNA controls are shown. Mean number of objects (nuclei) per well was used as a measure of proliferation, toxicity or cytostasis. Graphs represent the mean and standard error of mean number of objects from triplicate wells in 3 independent experiments expressed as a percentage of the initial seeding density (3000 cells). 100–150% was taken as no growth (cytostasis), <100% was taken as toxic. Inflammatory signalling was assessed using Mesoscale Discovery chemiluminescent ELISA (g). Hits were classed as ≥ 2/3 wells in ≥ 2/3 independent experiments passing the cut-off of mean scrambled control + 3 standard deviations. Values in heat-map boxes represent the number of repeat experiments in which at least 2/3 siRNA gave results greater than scrambled mean + 3SD.</p

siRNA selected for further investigation by priority group and confidence.

<p>Confidence ranking for nuclear area was based on number of standard deviations above the mean of plate negative controls and the number of library siRNA passing each threshold.</p

Gene ontologies associated with senescence inducing siRNA targets.

<p>An enrichment analysis for the gene ontology (GO) processes most significantly associated with the top 40 siRNA target genes was carried out in GeneGo from Metacore. The pie chart represents the number of GO processes falling into each of the 9 main categories expressed as a percentage of the 50 most significantly enriched GO processes.</p