Additional file 1 of Systematic analysis of different degrees of haemolysis on miRNA levels in serum and serum-derived extracellular vesicles from dogs

Additional file 1. Literature review of targeted miRNAs of this study. Human and dog-based studies indicating an association of miRNAs with haemolysis or suggesting their use as candidate biomarkers

Additional file 2 of Systematic analysis of different degrees of haemolysis on miRNA levels in serum and serum-derived extracellular vesicles from dogs

Additional file 2: Table 1. lists all primer sequences of selected miRNAs. Table 2. lists the synthetic RNA sequences of spiked miRNAs. Table 3. lists the synthetic cDNA sequences used for constructing miRNA-specific qPCR standard curves

ANXA8 expression arrests Kim2A8 cells in G₀.

<p>(<b>A</b>) Kim2A8 and Kim2RTS cells were grown in 6-well plates with or without 100ng/ml of dox for 48 hours. The graph shows the average percentage numbers of cells in G₀/G₁, S and G₂/M as quantified by FACS from three independent experiments. (<b>B</b>) Kim2A8 and Kim2RTS cells grown in chamber slides with or without 100ng/ml dox for six days were fixed and stained for Ki67 antigen. EGFP was used as a reporter of ANXA8 expression. (<b>C</b>) Graph showing the percentage of Ki67-positivity in the EGFP positive and negative populations of Kim2A8 cells grown with or without 100ng/ml dox. At least 1000 cells were analyzed in each population. (<b>D</b>) Western blot showing Ki67 and ANXA8 protein expression in cells after six days in culture. Actin was used as a loading control. Numbers show the relative intensities of ANXA8 and Ki67 bands respectively (normalised to actin) determined by measuring area pixel intensities using AIDA Image Analyzer software. The reduction of Ki67 levels (∼50%) is consistent with the reduced number of Ki67+ve Dox-treated Kim2A8 cells seen in (<b>B</b>).</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 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

ANXA8 is expressed strongest during pre-puberty.

<p>(<b>A</b>) Microarray results from a previous microarray study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119718#pone.0119718.ref041" target="_blank">41</a>], using RNA extracted from 3-, 4-, 5-, 6- and 7-week old CD1 mice, show a reduction in <i>AnxA8</i> mRNA at the onset of puberty. Signal intensities for two independent probes targeting <i>AnxA8</i> were normalised to cytokeratin 18 (Krt18) to eliminate changes due to differences in epithelial content. (<b>B</b>) qRT-PCR results for <i>AnxA8</i> normalised to Krt18 expression from RNA extracted from mammary glands of 3-, 4-, 6-, and 12-week-old mice. (<b>C-D</b>) Immunohistochemical analysis of ANXA8 expression using the E2R6.2 antibody on mammary glands from 3- (C) and 6-week old (<b>D</b>) mice showing staining for ANXA8 in the pre-pubertal rudiment and in ducts, but not TEB, of pubertal mice. Negative control (−ve ctrl): no primary antibody. Bars represent 100μm.</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 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