Co-localisation between internalised occludin and markers of the biosynthetic secretory pathway.

<p>A confluent monolayer of MDCK cells transiently expressing NPY-mRFP were fixed and immuno-localisation with mouse anti-occludin (1∶100) was performed. Cells were imaged by confocal microscopy. (A) Confocal image showing co-localisation between occludin and NPY-mRFP. (B) Quantification of 3 repeats of the experiments shown in (A) with a minimum of 13 cells analysed of each condition. *** p≤0.001.</p

Model (red lines) comparison with experimental data (blue lines).

<p>The grey spreads illustrate the model output obtained using 50 random values of parameters from the posterior distribution. The model consists of two variables: <i>M(t)</i> (membrane occludin) and <i>I(t)</i> (internal occludin). Occludin is assumed to undergo endocytosis, recycling and degradation at rates <i>η(t), ρ(t)</i> and <i>γ</i> respectively. The resulting equations are <i>dM/dt = −η(t)M+ρ(t)I, dI/dt = η(t)M−(ρ(t)+γ)I</i>. <i>η</i> and <i>ρ</i> are taken to be time-dependent following our finding that a linear version of the model cannot fit the data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111176#pone-0111176-g008" target="_blank">Figure 8</a>). We conclude that the experimental procedure that cells be cooled negatively impacts on these processes, which eventually return to maximal rates during the recovery period when cells are warmed. To represent this we use logistic functions: <i>η(t) = η_maxη₀e^rt/(η_max+η₀(e^rt−1))</i>, and likewise for <i>ρ(t).</i><i>η₀</i> and <i>ρ₀</i> represent the rates of endocytosis and recycling immediately following cooling and are assumed to be a fraction of the maximal rates, i.e. <i>η₀ = αη_max</i> and <i>ρ₀ = αρ_max</i> where 0<<i>α<1</i> and <i>r</i> is the rate of recovery of these processes. (A) and (B) correspond with the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111176#pone-0111176-g001" target="_blank">Figure 1B</a> (steady-state endocytosis and degradative trafficking of occludin) and (C) and (D) to those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111176#pone-0111176-g003" target="_blank">Figure 3B</a> (endocytic occludin recycling). In (A) and (B) all occludin is initially on the membrane (<i>M(0) = 100, I(0) = 0</i>) and in (B) degradation is inhibited via addition of BafA, hence <i>γ = 0</i>. In (C) and (D) all occludin is initially internal (<i>M(0) = 0, I(0) = 100</i>). Note that, to correspond with our experimental data, in (C) total occludin is plotted (i.e. membrane and internal occludin), while in (D) only internal occludin is shown. Parameters are estimated to be <i>η_max = 4.64(±2.1)</i> min⁻¹, <i>ρ_max = 3.05(±1.56)</i> min⁻¹, <i>r = 0.12(±0.15)</i> min⁻¹, <i>α = 0.02(±0.05)</i> and <i>γ = 0.013(±0.002)</i> min⁻¹, where the standard deviations are calculated from the marginal posterior distributions for each parameter.</p

A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes-2

Ioinformatics models as all genes examined were up regulated or specific to HUVECs and/or HDMECs.<p><b>Copyright information:</b></p><p>Taken from "A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes"</p><p>http://www.biomedcentral.com/1471-2164/9/153</p><p>BMC Genomics 2008;9():153-153.</p><p>Published online 7 Apr 2008</p><p>PMCID:PMC2346479.</p><p></p

Inhibition of protein synthesis reduces the amount of plasma membrane localised occludin.

<p>Serum starved MDCK cells were incubated with serum free DMEM plus DMSO (control) or 10 µM CHX for varying time-points. Cell surface proteins were then biotinylated and pulled down with NeutrAvidin beads. Plasma membrane abundance of occludin was quantified by Western blot analysis. (A) Incubation with CHX reduces both whole cell lysate and plasma membrane occludin levels. (B) Quantification of 3 independent experiments measuring whole cell lysate levels of occludin 4 hours post-CHX treatment normalised to Time 0. (C) Quantification of 3 independent experiments measuring plasma membrane levels of occludin 4 hours post-CHX treatment normalised to Time 0. p≤0.05 and ** p≤0.01.</p

Linear model (red lines) comparison with the experimental data (blue lines).

<p>The grey spreads illustrate the model output obtained using 50 random values of parameters from the posterior distribution. (A) and (B) use data taken from the steady-state endocytosis and degradative trafficking of occludin experiment (<i>M(0) = 100, I(0) = 0</i>), see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111176#pone-0111176-g001" target="_blank">Figure 1B</a>, both representing internal occludin with no degradation occurring in (B) (<i>γ = 0</i>). Data in (C) and (D) are both obtained from the endocytic occludin recycling experiment (<i>M(0) = 0, I(0) = 100</i>), see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111176#pone-0111176-g003" target="_blank">Figure 3B</a>, the first being the total occludin (<i>M(t)+I(t)</i>), and the second internal occludin. The model equations are given by <i>dM/dt = −ηM+ρI, dI/dt = ηM−(ρ+γ)I</i>, i.e. the only change from the model in the main text is that the rates of endocytosis and recycling are constant. For all high likelihood parameter values, the initial rise of internalized occludin in the trafficking experiments is considerably faster than experimentally observed. This suggests the necessity of a slower time scale, which is included in the main model as the rate of recovery from cooling.</p

Diagram demonstrating occludin trafficking under steady-state conditions.

<p>During the initial stages of MDCK cell polarisation occludin undergoes endocytosis followed by lysosomal degradation and low-level recycling. This is coupled to occludin biosynthesis in order to maintain plasma membrane occludin homeostasis.</p

Co-localisation between internalised occludin and recycling endosome markers.

<p>A confluent monolayer of MDCK cells transiently expressing Rab11-GFP were fixed and immuno-localisation with mouse anti-occludin (1∶100) was performed. Cells were imaged by confocal microscopy. (A) Confocal image showing co-localisation between occludin and Rab11-GFP. (B) Quantification of 3 repeats of the experiments shown in (A) with a minimum of 12 cells analysed of each condition. *** p≤0.001.</p

Steady-state endocytosis and degradative trafficking of occludin.

<p>A confluent monolayer of MDCK cells were serum starved in the presence of either DMSO or 250 nm BafA. Cell surface proteins were biotinylated and trafficking allowed to recommence for designated time-points in the presence of DMSO or BafA. Cell surface biotinylated proteins were reduced, thus only internalised proteins remain biotinylated. Biotinylated proteins were pulled down with NeutrAvidin beads. Protein abundance was quantified by western blot analysis. (A) Treatment with BafA (inhibitor of lysosomal degradation), attenuates the decrease in occludin signal following endocytosis observed in DMSO treated control cells. (B) Quantification of 3 repeats of the experiments shown in (A). (C) Quantification of the decrease in occludin signal between 30 and 120 minutes comparing control to BafA treatment. * p≤0.05.</p

A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes-1

Bioinformatics models as all genes examined were up regulated or specific to HUVECs and/or HDMECs.<p><b>Copyright information:</b></p><p>Taken from "A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes"</p><p>http://www.biomedcentral.com/1471-2164/9/153</p><p>BMC Genomics 2008;9():153-153.</p><p>Published online 7 Apr 2008</p><p>PMCID:PMC2346479.</p><p></p

Co-localisation between internalised occludin and late endosome/lysosomal compartments increases after BafA treatment.

<p>Incubation with BafA increases co-localisation between occludin and CD63-GFP in comparison to control treated cells. A confluent monolayer of MDCK cells transiently expressing CD63-GFP were incubated with DMSO or 250 nm BafA for 2 hours prior to fixation and immuno-localisation with mouse anti-occludin (1∶100). Cells were imaged by confocal microscopy. (A) Confocal image showing co-localisation between occludin and CD63-GFP in the presence of DMSO or BafA. (B) Quantification of 3 repeats of the experiments shown in (A) with a minimum of 19 cells analysed of each condition. *** p≤0.001.</p