Disorder profile of the cytoplasmic parts of 263 single-pass transmembrane proteins with long cytoplasmic tails.

<p>(>150 residues). Standard errors of the mean values are displayed in yellow.</p

Evolutionarily conserved motif region in the Synaptotagmin 14 linker region.

<p>The upper panel shows a graphical representation of whole Synaptotagmin 14 protein. The transmembrane region (TM), the linker and the two C2 domains are shown. The lower panel shows the first 120 residues of an alignment of synaptotagmin 14 with identified orthologues. Red bars indicate Relative Local Conservation (RLC) and the blue bars indicate IUPred disorder scores. The brown background shows an ordered part (IUPred score ><0.3) around the transmembrane region.</p

Frequency of conserved motifs.

<p>The frequency (blue line) depicts the number of instances that a specific residue position falls inside a predicted motif. Disorder profile of single-pass transmembrane proteins when the membrane region was included in the analysis is shown in red, with standard errors displayed in yellow. The number of proteins that have a residue at a specific position is shown with a black line.</p

Conserved motif density in long (>150 residues) cytoplasmic tails of single-pass transmembrane.

<p>Conserved motifs density is defined as the number of conserved motifs that span a residue divided by the number of residues that are disordered.</p

Disorder profile of single-pass proteins.

<p>Mean IUPred disorder profile (in red) for residues from 803 human single-pass transmembrane proteins, with respect to distance from the membrane (position 0; extracellular negative, intracellular positive). The membrane region was included in the analysis, but has been omitted from the graph. Standard errors are displayed in yellow. The number of proteins that have a residue at a specific position is shown with a black line. TM: transmembrane.</p

Amino acid composition.

<p>Standard error bars plotted on the graph are very close to the mean values. EC: extracellular. IC: intracellular.</p

ANXA8 expression induces morphological changes in Kim-2 cells.

<p>Kim2A8 and Kim2RTS cells were grown in the presence or absence of 100ng/ml dox. Pictures were taken after 48 hours (<b>A</b>) or six days (<b>B</b>) of treatment. EGFP was used as a reporter of ANXA8 expression. Both proteins are expressed from opposite sides of a bidirectional promoter. (<b>C</b>) Nuclear sizes were analysed after 6 days by measuring the nuclear area (stained with DAPI) of at least 90 individual cells from each dox-treated and untreated populations using ImageJ. There was a significant difference between Kim2A8 cells expressing and not expressing ANXA8 (ANOVA: p<0.05).</p

ANXA8 positive cells are ERα and Ki67 negative.

<p>Co-immunofluorescence staining for ANXA8 and Ki67 (<b>A</b>) or ERα (<b>C</b>) in 6-week old mice shows that those cells strongly positive for ANXA8 are negative for ERα and Ki67. Graphs represent the mean percentage of ANXA8 positive and negative cells that are positive or negative for Ki67 (B) or ERα (D) based on 1,000 cells per developmental time point (<b>B</b>) and at least 500 cells (<b>D</b>). V6: virgin 6 weeks; P4.5: pregnancy day 4.5; P12.5: pregnancy day 12.5. Error bars denote standard error of the mean.</p

ANXA8 expression inhibits proliferation of Kim2A8 cells.

<p>(<b>A</b>) Kim2A8 and Kim2RTS cells were seeded in 24-well plates, allowed to attach and grown in the presence or absence of 100ng/ml dox (first treatment at time point 0). At each time point protein extracts were prepared (in duplicates). The graph shows the amount of protein as determined by BCA assay against time. This assay was performed in triplicate and the graph shows a representative result from one experiment. (<b>B</b>) Cells were seeded in 96-well plates and treated with dox for 48 hours, labelled with BrdU and the incorporation of BrdU was quantified and plotted for each condition (six wells per condition). ***p< 0.001 (<b>C</b>) Equal amount of cells (250–300) were grown in the presence or absence of 100ng/ml dox. After 14 days cells were fixed, stained and the plates photographed. A representative plate per condition is shown. The experiment was carried out in triplicate. (<b>D</b>) Graph showing the number of colonies per plate from the experiment (<b>C</b>) as quantified by Image J (**p<0.003).</p

ANXA8 is expressed in ERα−ve/c-kit+ve luminal progenitor cells.

<p>(<b>A</b>) Primary mammary epithelial cells were sorted and RNA extracted as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119718#pone.0119718.ref039" target="_blank">39</a>]. qRT-PCR was used to measure <i>AnxA8</i> mRNA abundance in four different populations. <i>AnxA8</i> was not detected in either mammary stem cells (MaSC) or myoepithelial cells. Although very low levels were detected in differentiated ERα+ve luminal epithelial cells, ERα−ve luminal epithelial progenitor cells showed a 17-fold higher abundance. The graph shows the abundance relative to the levels of expression in differentiated cells and 95% confidence limits. (<b>B</b>) Bar graph showing the proportion of c-kit+ve/AnxA8+ve and c-kit+ve/AnxA8−ve cells during puberty (V6), early (P4.5) and late (P12.5) pregnancy. 1,000 cells were assessed per developmental time point. Error Bars denote standard error of the mean. (<b>C</b>) Co-immunofluorescence staining for ANXA8 and c-kit in mouse mammary gland from an early-pregnant (day 4.5) mouse.</p